Epithelia Suspended in Collagen Gels Can Lose Characteristics of Migrating Mesenchymal Cells Polarity and Express

This study of epithelial-mesenchymal transformation and epithelial cell polarity in vitro reveals that environmental condit ions can have a profound effect on the epithelial phenotype, cell shape, and polarity as expressed by the presence of apical and basal surfaces. A number of different adult and embryonic epithelia were suspended wi th in native collagen gels. Under these condit ions, cells elongate, detach from the explants, and migrate as individual cells wi th in the three-dimensional lattice, a previously unknown property of well-dif ferentiated epithelia. Epithelial cells from adult and embryonic anterior lens were studied in detail. Elongated cells derived from the apical surface develop pseudopodia and f i lopodia characteristic of migratory cells and acquire a morphology and ultrastructure virtual ly indist inguishable from that of mesenchymal cells in vivo. It is concluded from these experiments that the three-dimensional collagen gel can promote dissociation, migration, and acquisition of secretory organelles by differentiated epithelial cells, and can abolish the apical-basal cell polarity characteristic of the original epithel ium. Epithelium is the tissue that lines body surfaces. The cells are polarized with respect to the outside or free surface, with Golgi zones usually in the apical cytoplasm. Specializations such as microvilli and junct ional complexes distinguish the apical and lateral surfaces from the basal surface, which is attached to extracellular matrix in the form of a basal lamina (13). In the early embryo, certain epithelia give rise to mesenchymal cells that invade extracellular matrices and lose the apical, lateral, and basal specializations that characterized the parent epithelium. Their at tenuated cell bodies and bipolar or stellate shapes distinguish them from closely apposed, polygonal epithelial morphology, as does the ability to migrate with a three-dimensional extracellular matrix. The epithelial and mesenchymal phenotypes, once formed, appear to be stable. If epithelial cells are grown on top of collagenous matrices in vitro, they develop a flat basal surface next to the matrix (14-16), whereas mesenchymal cells (16) and presumptive mesenchymal cells (3) invade underlying extracellular matrix. Adult thyroid (7) and normal and malignant mammary epithelial cells (20), embedded within collagenous matrices in vitro, organize into polarized epithelial structures with basal surfaces contacting the collagen gel and apical poles facing a central cavity. Cells cloned from a mammary tumor cell line, however, can form pointed outgrowths of loosely associated elongated cells that invade into collagen gels from surface monolayers (2). To study further the stability of the epithelial phenotype, especially as regards surface polarity, we suspended a number of different epithelial tissues within gelling solutions of collaTHE IOURNAL OF CELL BIOLOGY VOLUME 95 OCTOBER 1982 333-339 © The Rockefeller University Press • 0021-9525/82/10/0333/07 $1.00 gen. To our surprise, we found that adult lens epithelium and a number of embryonic epithelia that do not form mesenchyme in vivo routinely give rise to cells that separate from the tissue explant and migrate individually into the surrounding collagen gel. The migrating cells assume a bipolar morphology and free structure almost indistinguishable from that of mesenchymal cells in collagen gels (1, 19). In this paper, we report the behavior of corneal, notochordal, limb, and lens epithelia, and endothelial cells, suspended in collagen gels, and describe in more detail the behavior in gels of adult and embryonic anterior lens epithelium, which can be isolated as a pure population of simple cuboidal epithelial cells resting on an intact basal lamina. MATERIALS AND METHODS Collagen Gel Preparation Type I collagen was extracted from adult rat tail tendon with acetic acid and used to prepare native collagen gels by a modification of the method of Elsdale and Bard (9) using Ham's F-12 medium (Gibeo Laboratories, Grand Island Biological Co., Grand Island, NY). CoLlagen was first extensively dialyzed against Yl0 F-12 and then diluted to 1.5 mg/ml. Then, 1.4 ml was precipitated at 4°C with 0.2 ml F-12 (101 mg/ml), 0.2 ml sodium bicarbonate (11.76 mg/ml), and 0.2 ml fetal calf serum (FCS, Flow Laboratories, Rockville, MD). Next, 0.3-0.5 ml of this collagen solution (final concentration 1 mg/ml) was pipetted onto plastic tissue culture dishes (Falcon Labware, Div. Becton, Dickinson and Co., Oxnard, CA), to form a drop I cm in diameter. The tissue was quickly suspended within the gelling solution and the gel was incubated for 15 min at 37°C before addition of Ham's F12 medium supplemented with 10% FCS, 10 mM glutamine, 2.5 gg/ml fungizone (Gibco Laboratories) and 50 ~g/ml gentamycin (Schering, Kenilworth, N J). 333 on O cber 9, 2017 jcb.rress.org D ow nladed fom Epithelia were also cultured on the upper surface of collagen gels prepared by pipetting 0.5 ml of gelling solution (see above) onto the tissue culture dish and allowing it to polymerize at 37°C for 30 rain. The intact lens epithelium isolated as described below was placed on the surface of the gel, incubated 1-2 h (37°C) in a small volume of medium, then covered with complete medium for the duration of the culture period. Cell and Tissue Isolation Notochords were isolated from posterior trunk of 2~1-old (stage 13) chick embryos with 1% trypsin (Difco Laboratories, Detroit, MI) in HBSS. There was no mesenchymal cell contamination because no mesenchyme is present in the posterior trunk region (6, 8). Limb bud ectoderm was isolated from 5-d-old chick embryos by the method of Erick and Saunders (10), which permits epithelium, to. be separated completely from limb mesoderm. Corneal epithelium was isolated with trypsin/collagenase as reported by Meier and Hay (15), which routinely yields pure epithelial tissue. Both embryonic and adult avian lens epithelia were dissected with lens capsnie intact as pieces 1 mm in diameter from the anterior portion of the lens by the method of Piatigorsky (17). Embryonic avian lens cells were also dissociated from the lens capsule by incubating tissue fragments in a solution of 0.2% collagenase (Sigma Chemical Co., St. Louis, MO) in HBSS for 30 min followed by 0.05% trypsin (type I, Sigma Chemical Co.) plus 0.02% EDTA in HBSS without Ca + and Mg ++ for 15 min at 37"C. The dissociated cells were centrifuged for 5 rain, washed in complete medium and repelleted. This pellet was then pipetted into the gelling collagen. Corneal endothelial cells were isolated from adult bovine eyes and maintained in Dulbecco's modified Eagle's medium H-16 (DME) supplemented with 10% FCS, 10 mM glutamine, 2.5 ~g/ml fungizone, 50/~g/ml geutamycin, and 100 rig/ ml purified brain fibroblast growth factor (FGF, added every other day; a gift from Dr. D. Gospodarowicz, University of California, San Francisco), as previously described (11, 12). Cultures were passaged every week with a split ratio of l to 64, and cells were used between the 5th and 30th generations. Cells were suspended in collagen gels by first dissociating them from the plastic dish with trypsin-EDTA in HBSS without Ca + and Mg ++, centrifuging in complete DME medium and pipetting the pellet into the gelling coliagen, Cultures were maintained in complete medium without FGF. Light and Electron Microscopy Live cultures of tissues suspended within collagen gels were observed and photographed with inverted phase-contrast and Nomarski microscopes. For transmission electron microscopy (TEM), tissue or cuRures were flied for 30 min in 2% paraformaldehyde and 2.5% glutaraldehyde in cacodylate buffer (0.1 M and posffixed in 1% osmium tetroxide in 0.1 M cacodylate buffer, pH 7.4, at 4°C for 30 rain. They were stained en bloc in 1% uranyl aerate, dehydrated, and embedded in Spurt (D.E.R. 736 embedding kit, Tousimis Research Co., Rockville, MD). Thin sections were cut on a Sorvall MT2-B ultramicrotome (Dupont Instruments-Sorvall Biomedical Div., Newtown, CT) and stained with lead citrate (0.2%); thick sections were stained with 1% tolnidine blue in 1% sodium borate.