Proliferative and differentiative response of corneal and limbal epithelium to extracellular calcium in serum-free clonal cultures.
An increasing concentration of extracellular Ca2+ ([Ca2+]e) consistently induces epithelial differentiation, but its effect on proliferation remains variable. We investigated the effect of [Ca2+]e on two different cell populations: the peripheral corneal (PC) and limbal (L) epithelia, the latter containing corneal stem cells. Primary clonal (18 cells/cm2) cultures from rabbit limbal and peripheral corneal epithelia were established in serum-free MCDB 151 medium containing growth-promoting agents and 0.03, 0.3, or 1.8 mM Ca2+. During early culture life, colony size and the BrdU labelling-index of L and PC, assayed on day 6, increased in response to increasing [Ca2+]e; cell attachment and colony-forming efficiency remained unchanged for both L and PC epithelia. These results indicate that increasing [Ca2+]e, under these defined conditions, stimulates the proliferation of transient amplifying cells, but does not stimulate the differentiation of stem cells into clonal proliferation. A 10-fold increase of the seeding density or prolongation of the culture up to day 14 or 21 changed the response to [Ca2+]e allowing better proliferation in lower [Ca2+]e. Only cells grown as a monolayer in 0.03 mM Ca2+ could still be passaged on day 14, whereas cells in higher [Ca2+]e showed increasing stratification and cell detachment and could not be passaged. Normal cellular differentiation accessed by the expression of a cornea-type K3 keratin, recognized by the monoclonal antibody AE-5, was enhanced by increasing [Ca2+]e. Abnormal differentiation featured by the formation of cornified envelopes was only observed in higher [Ca2+]e. These results indicate that [Ca2+]e promotes the proliferation of relatively undifferentiated transient amplifying cells under clonal, serum-free culture conditions. Factors that enhance differentiation, such as seeding density or prolonged culture life, can modify this response and allow better proliferation in low [Ca2+]e.
Kruse
F E
FE
Department of Ophthalmology, Bascom Palmer Eye Institute, University of Miami School of Medicine, Florida 33101.
Tseng
S C
SC
D000818:Animals; D001973:Bromodeoxyuridine / Q000493:pharmacokinetics; D002118:Calcium / Q000378:metabolism*; D002454:Cell Differentiation; D002455:Cell Division; D002478:Cells, Cultured; D004728:Endothelium, Corneal / Q000166:cytology* / Q000378:metabolism; D005110:Extracellular Space / Q000378:metabolism*; D016850:Limbus Corneae / Q000166:cytology* / Q000378:metabolism; D009994:Osmolar Concentration; D013997:Time Factors
D001973:Bromodeoxyuridine; D002118:Calcium