Differentiation of Normal Human Mammary Epithelial Cells in Culture: An Ultrastructural Study ' JOSE RUSSO, PHILIP FURMANSKI, RONALD BRADLEY, PETER WELLS AND MARVIN A. RICH Department of Biology, Michigan Cancer Foundation 110 East Warren Avenue, Detroit, Michigan 48201
ABSTRACT An ultrastructural and cytochemical study of normal human mammary epithelial cells cultured from post-weaning breast fluids is described. Cells were examined at the time of plating and at intervals up to 28 days in culture. Three different stages in the morphological differentiation of these cells in vitro were observed: (1) the first stage was the formation of a monolayer of single cells, which occurred between days 1 and 10 in culture. The cells in this stage were not interconnected by junctional complexes and lacked Mg++dependent ATPase activity in the plasma membranes, but did contain a large quantity of lipid and exhibited some secretory characteristics. ( 2 ) The second stage, occurring at 10 to 16 days in culture, was characterized by the formation of junctional complexes, the appearance of Mg++-dependent ATPase in the plasma membrane and a decrease in the number of dense bodies with peroxidase activity. ( 3 ) The third stage, occurring at 16 to 28 days in culture, was characterized by the formation of stratified layers of epithelial cells, which were interconnected by a large number of desmosomes with numerous pleomorphic microfilaments. The Mg++-dependentATPase activity in the plasma membrane was retained and the dense bodies with peroxidase activity were rarely observed at this stage. During the last seven days in culture, secondary lysosomes, autophagosomes and residual bodies were prominent in the cells of the stratified layer. After 28 days in culture, the cells began to round up and slough off the culture plate.
We have previously reported that epithelial cells can be isolated from postweaning breast fluids of normal human donors (Furmanski et al., '74a,b). In the presence of autologous serum, these cells will grow in culture with a generation time of 60-72 hours. Morphological studies suggest that these cells are derived from the mammary epithelium, shed into the fluid as a consequence of the normal process of involution following the termination of active lactation (Russo et al., '75). We have observed that these cultures of normal human mammary epithelial cells progress through a distinct series of morphological changes. These changes, which suggest a pattern of cell association and differentiation of mammary epithelium in vitro, are detailed here. MATERIALS AND METHODS
Post-weaning breast fluids obtained from AM. J. ANAT., 145: 57-78.
normal human donors were centrifuged at low speed (200 x g ) . The resulting pellet of cells was resuspended in culture medium and the number of cells determined by hemacytometer count. The culture medium consisted of Dulbecco's modified Eagle's medium containing autologous serum ( 15% v/v), insulin ( 10 pg/ml) and antibiotics, as described previously (Furmanski et al., '74a,b). The cells attached to the bottom of the culture chamber and grew with a doubling time of 6072 hours. Cells from 6, 10, 14, 16, 24 and 28 days after plating were used in this study. Cells isolated from the post-weaning fluids were also examined prior to the initial plating. Accepted October 7, '75. 1 This work was supported by Contract NOI-CP33347 and Grant CA 14100 from the National Cancer Institute and an institutional grant to the Michigan Cancer Foundation from the United Foundation of Detroit, Michigan.
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The cultures were fixed in 1.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2, for one hour at 4°C. After this initial fixation, the plate was divided into four portions while immersed in cold buffer. One portion of each plate was fixed for two hours at 25°C according to the method of Hirsch and Fedorko ('68) for morphological studies. After fixation, the material was washed in 0.85% NaC1, treated with uranyl acetate, and dehydrated through graded concentrations of ethanol. The monolayers of cells were removed from the plastic plate with propylene oxide, and infiltrated and embedded using Luft's Epon ('61) in a ratio of 1 part mixture A to 1 part mixture B. The other portions of the plate were processed for the cytochemical localization of Mg++dependent ATPase, peroxidase, and alkaline phosphatase. These pieces of the culture plate, which had been fixed for one hour before the plate was divided, were transferred to fresh fixative containing 1.5% glutaraldehyde in 0.1 M cacodylate with 1% sucrose, pH 7.2, and fixed for an additional hour at 4°C. After fixation, the portions of the plate were washed in 0.1 M cacodylate with 6% sucrose at 4°C.
A small sample of cells isolated directly from post-weaning fluids was removed prior to the initial plating and fixed by suspension in 1.5% glutaraldehyde for two hours at 4°C. After fixation, the cells were pelleted in a Beckman Microfuge and washed by resuspension in cold buffer. All cytochemical material was held at 4°C in the last buffer wash until the appropriate substrate mixtures were added. Enzyme substrates were used immediately after preparation. The Mg++-dependent ATPase cytochemical reaction was performed using the incubation mixture described by Wachstein and Meisel ('57) and incubated at 37°C for 20 minutes. The cytochemical localization of peroxidase was accomplished using the incubation medium described by Graham and Karnovsky ('66) and incubated for 20 minutes at room temperature. The alkaline phosphatase reaction was performed using the method of Mayahara and Ogawa ('68), and incubated at 37°C for 60 minutes. Material was incubated simultaneously
using control media for each of these enzyme reactions. Control media were prepared by replacing the appropriate cytochemical substrate with an equal volume of distilled water. After incubation, the reaction mixtures were drawn off and the cells or monolayers were washed in 0.1 M cacodylate with 6 % sucrose at 4°C. The cells were postfixed in 1% osmium tetroxide in 0.1 M cacodylate for two hours, washed with buffer and dehydrated using ethanol. The monolayer of cells was removed from the plastic plate with propylene oxide. All material for cytochemical study was infiltrated and embedded using an EponAraldite mixture (Mollenhauer, '64). Thin sections were cut for electron microscopy using a diamond knife mounted in an LKB Ultrotome and examined with either a Siemens Elmiskop 1A at 60 kv or with a Hitachi HU-1lA at 75 kv. The majority of sections that were observed and photographed were stained with 2% uranyl acetate and Reynold's lead citrate ('63). However, some sections from the cytochemical blocks were stained only with 2% uranyl acetate in order to evaluate the histochemical localizations without the effect of lead staining. RESULTS
In cultures of normal human mammary epithelial cells obtained from post-weaning fluids, three distinct stages of morphological differentiation of the cells were observed during the 28-day culture period. The first stage, which occurred during the first ten days in culture, consisted of a monolayer of single cells in which no junctional complexes were observed. The second stage, observed during the tenth to sixteenth day, consisted of clusters of four to eight epithelial cells which were connected by desmosomes and junctional complexes. The third stage, from 16 to 28 days, was characterized by stratified layers of epithelial cells with large numbers of desmosomes and tight junctions interconnecting the cells. Cells f r o m post-weaning fluid The epithelial cells obtained directly from post-weaning fluids possessed a high concentration of lipid droplets, with few
NORMAL HUMAN MAMMARY EPITHELIAL CELLS
mitochondria, some secretory granules, and a poorly developed endoplasmic reticulum (figs. 1, 2, 9). The cell surface had numerous microvilli of varying size and shape, The nuclei of these cells had a regular shape with some indentations, typical of the nuclei of normal mammary epithelium (Tannebaum et al., '69). One or two nucleoli were usually present in each nucleus (figs. 1, 2 ) . The cells obtained directly from postweaning fluids did not show any reaction product in their plasma membranes for Mg++-dependentATPase (fig. 9 ) or alkaline phosphatase (fig. 1 ) . However, they did show a small number of bodies with peroxidase activity (fig. 2 ) .
The monolayer During the first ten days in culture, two cellular types were observed in the monolayer. The first was a flat cell (fig. 3 ) with a small number of elongated microvilli and few lipid droplets. The Golgi complex was located in a supra-nuclear position. The nuclei were generally ovoid i n shape and located in the basal portion of the cells. Heterochromatic material was scarce and was attached to the inner membrane of the nuclear envelope. The second type of cell was more spherical or elevated in nature (figs. 5, 7 ) . These cells had a larger number of microvilli on their surface and possessed a higher concentration of lipid droplets i n their cytoplasm. The Golgi complex was well developed and usually found in the apical portion between the free border of the cell and the nucleus, which resembles the polarity of glandular epithelium (fig. 7). Some granules containing electron dense material were observed in proximity to the Golgi complex (fig. 8 ) . Multivesicular bodies were observed frequently at this stage (fig. 6 ) and lipid droplets were often seen apparently fusing with lysosomes to form lipolysosomes (fig. 8). Rough endoplasmic reticulum was present (fig. 5 ) and free ribosomes were dispersed throughout the cytoplasm. The nuclei of these cells were relatively spherical and were frequently observed in the basal portion, but rarely in the apical portion of these cells (fig. 7 ) . Nucleoli were found either in the
59
center of the nucleus or associated with the nuclear envelope (figs. 5, 7). Both types of cells became firmly attached to the petri dish and a palisade of thin microfilaments 5-7 mp in diameter (figs. 20, 21) and granular material was found adjacent to the attached surface of these cells (fig. 3 ) . Although the cells in the monolayer were connected by interdigitations (fig. 5), typical junctional complexes were not observed. The plasma membranes of these cells did not exhibit Mg' +-dependent ATPase activity. Toward the end of this stage, the cells in the monolayer came into contact with each other and began to form multicellular clusters (fig. 10). Mg'+-dependent ATPase activity was detected in the plasma membranes of the interdigitations that occurred between these cells (figs. 10, 12). The flat cells of the monolayer possessed a number of dense bodies with peroxidase activity as demonstrated by incubation with 3,3' diaminobenzidine (figs. 3, 4 ) . The plasma membranes of the monolayer cells were negative for alkaline phosphatase activity, a s were the plasma membranes of the cells obtained directly from post-weaning fluids.
Multicellulnr clusters Between 10 and 16 days in culture, the confluent monolayers were characterized by well developed junctional complexes (Russo et al., '75). Scattered among the monolayers were clusters of four to ten cells that were joined together by numerous desmosomes (fig. 14). Cell surfaces showed a number of short and blunt microvilli. Mitochondria were scattered throughout the cytoplasm and were polymorphic with many transverse cristae. The rough endoplasmic reticulum was poorly developed but was more abundant than the smooth endoplasmic reticulum. Few inclusions and a decreased number of lysosomes were observed i n these cells. A distinct feature was the appearance of tonofilaments which were associated with the terminal web of the desmosomes (figs. 17, 18). The nuclei of the cells were found in the apical portion and were polymorphic, with numerous indentations. Scarce heterochromatic material and usually one nucleolus were seen (fig. 14). Nuclei in the
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basal portion of the cluster were more elongated with an even distribution of heterochromatic material. Dense bodies, positive for the 3,3' diaminobenzidine reaction, were absent. Mg++-dependent ATPase was present on the cell surface and between the cells. The alkaline phosphatase cytochemical reaction was negative, as in the preceding stages. Stratified layers The formation of clusters was followed by the growth of cells in stratified layers, occurring generally after 16 days in culture and persisting until the cells rounded up and sloughed off the culture plate. The stratification consisted of two to six layers of cells joined together by numerous desmosomes (figs. 15, 16). These cells were flatter than those observed in the clusters described above. In some of these structures, cells with morphological characteristics very similar to those observed during the first week in culture were observed in the basal strata (fig. 15). These cells exhibited numerous lipid droplets and dense bodies with peroxidase activity, suggesting that they had originated from the monolayer cells by cell division and morphological differentiation. Only a few short and blunt microvilli were observed at the cell surfaces (figs. 17, 19, 26). The most prominent feature of the cytoplasm was the presence of numerous bundles of microfilaments (figs. 16, 19, 24). These filaments may be classified according to their diameter and distribution into three different types. The first type, 5-7 mp in diameter, was usually found in the basal portion of the cell adjacent to the surface that was attached to the culture vessel (figs. 20, 21). The second type of filament was found in bundles scattered throughout the cytoplasm (fig. 24) and these thick filaments were 14-15 mp in diameter (fig. 25). The third type was intermediate in diameter, 8-10 mp, and these filaments were observed in parallel arrays in the cytoplasm (figs. 22, 23) and adjacent to the nucleus (fig. 16). Tonofibrils were always found associated with the terminal web of the desmosomes (figs. 16-19). They appeared in long arrays originating from the desmosomes and extending into the cytoplasm.
Their diameters ranged from 7-10 mp and they were similar in size and appearance to the intermediate filaments described above. Numerous polymorphic mitochondria were scattered throughout the cytoplasm (figs. 19, 26). The Golgi complex was no longer visible in these cells. The rough endoplasmic reticulum was poorly developed (figs. 15, 24), and free ribosomes were scattered throughout the cytoplasm (figs. 17, 19). The nuclei were polymorphic and heterochromatic material was scarce or absent. Usually only one nucleolus was present (figs. 15, 16, 19, 26). Between 24 and 28 days in culture, several types of lysosomes appeared in the cells of the stratified layers. Secondary lysosomes, autophagosomes, and residual bodies became a prominent element and occupied a major portion of the cytoplasm (figs. 26, 28, 29). The multivesicular bodies (fig. 28) could be classified as secondary or autophagic lysosomes. The degenerative bodies or residual bodies were considered to be tertiary lysosomes in which indigestible cellular debris was concentrated during the final stages in culture (figs. 26, 29). Whorl-like or myelinlike structures were observed within the residual or degenerative bodies (figs. 26, 29). Crystal-like structures were often seen associated with lysosomes in the cytoplasm of these cells (fig. 27). During the final stages of culture, a strong Mg++-dependentATPase cytochemical reaction was observed on membranes between the cells (figs. 11, 13). However, in contrast to the monolayer cells previously described, the bodies with peroxidase activity were only rarely observed. Following four weeks in culture, the cells rounded up and sloughed off the surface of the culture vessel. DISCUSSION
Post-weaning fluids from normal human donors contain epithelial cells that can be grown in culture (Furmanski et al., '74a,b; Russo et al., '75). When these cells are plated, they attach to the bottom of the dish and grow to form confluent monolayers. One cell type observed in these cultures is seen only during the first ten days and
NORMAL HUMAN MAMMARY EPITHELIAL CELLS
in low concentrations. The large number of dense bodies with peroxidase activity and the elongated microvilli suggest that these cells are macrophages. Similar characteristics have been described for monocytes and macrophages in culture (Reaven and Axline, '73). Another type of cell observed during the first ten days in culture exhibits many features characteristic of breast epithelium, as previously described (Russo et al., '75). These cells possess a marked polarity demonstrated by microvilli on the free surface and the supranuclear position of the Golgi apparatus, which was associated with electron dense granules that resembled secretory products. The well developed Golgi apparatus, the rough endoplasmic reticulum and the increased quantity of lipid droplets which are present in these cells and in the post-weaning fluid cells have also been observed in lactating mammary epithelium (Wellings, '68). However, possibly due to the absence of hormones which are normaly present in the human breast during lactation, these cells in culture do not appear to release secretory products. Our observations suggest that the digestion of lipid droplets and the formation of lipolysosomes occur during this phase in culture, and that this process leads to the reduction of lipid that is observed in the later stages. Lipolysosomes have also been demonstrated to play a role in the process of cellular differentiation of the Leydig cells in the testes of the mouse (Russo, '71). During the second stage, from 10 to 16 days in culture, multilayered clusters of cells are formed which are the morphological precursors of the stratified layer. Most of the characteristics which were described for the monolayer are not observed in the clusters; the Golgi complex is diminished and the lipid droplets are much scarcer, Clusters of cells may be formed in a manner suggested by figure 10, in which the microvilli of these cells have come in contact and the cells are beginning to grow on top of one another. When the cells begin to join one another, Mg++dependent ATPase activity appears in the plasma membrane and, later, desmosomes and permanent junctions are formed.
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At the present time, the function of ATPase activity in the plasma membranes of lactating mammary epithelium is not completely understood. Localizations of ATPase activity in lactating mammary gland are not in agreement with biochemical and physiological data (Huang and Keenan, '72, Baumrucker and Keenan, '75, Linzell and Peaker, '71). Other authors have established that this enzyme is not directly involved in the process of lactose secretion (Vreeswijk, '75). However, we have demonstrated that Mg' +-activated ATPase is a permanent marker for epithelial cells in the resting mammary gland (Russo and Wells, '75a,b). Similar results have been reported in pathological conditions of the breast (Murad, '71). In mouse mammary gland, Pitelka ('73) observed that the desmosomes which are present in the resting gland disappear, and only tight and gap junctions remain during lactation. The lack of desmosomes and ATPase activity, in addition to the morphology of the post-weaning fluid cells and the first-stage cells in culture, suggest that these cells are in a secretory state. The correlation between the appearance of desmosomes and the detection of ATPase activity in the plasma membrane after ten days in culture suggests a transition from a secretory state to a resting state. The formation of stratified layers is one of the characteristics of these human breast cells in culture. Unlike those described by Kruse and Miedema ('65), these multilayered structures do not suggest characteristics of transformation in vitro but appear to reflect the cell type and the conditions of culture (Sanford, '61, '67). A characteristic of normal diploid cells is finite growth in culture. The capacity of normal cells to exhibit aging and senescence is a n important marker in differentiating normal from transformed cells. One of the criteria for aging is the presence of residual bodies, autophagolysosomes, and lipofucsin granules. The increase of these organelles with age has been described by Brunk ('73a,b). The accumulation of residual bodies, described as an indication of the finite lifespan of normal cells in culture (Brunk, '73a), was observed during the fourth week, just prior
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to the expiration of the cultures described in this report. The process of cellular degeneration, which occurs in these breast epithelial cells, differs from the processes of cellular degeneration and keratinization which occur in the epithelium of the epidermis (Flaxman et al., '67). The cytoplasmic filaments which were observed in the stratified layer cells have been grouped according to their diameter and morphology. The first class of filaments, which are 5-7 mp in diameter, has been observed in other types of cells in vitro and in vivo and has been shown to possess actin-like properties (Ishikawa et al., '69; Pollard et al., '70). The second class of filaments, with diameters of 1415 mp, has an appearance similar to the thick myofilaments which have been observed in smooth muscle cells (Devine and Somlyo, '71). The third class of filaments, with diameters of 8-10 mp, is oriented in bundles in the cytoplasm. Similar arrangements of filaments of this diameter have been found in fibroblasts and melanocytes (McNutt et al., '73; Jimbow and Fitzpatrick, '75). A massive accumulation of filaments in the cytoplasm has been related to the process of keratinization that is observed in the epithelium of the skin. This keratinization is also followed by other changes such as nuclear degeneration, the disappearance of ribosomes and mitochondria, and alterations of the desmosomes (Flaxman et al., '67, '72). These changes in cellular organelles were not observed in the human breast epithelial cells in culture, and evidence of keratohyaline or precursors of keratin were not detectable. Histochemical assays for plasma membrane alkaline phosphatase were negative in both the post-weaning fluid cells and the cells in all stages during culture. In the mammary gland, alkaline phosphatase has been shown to be a cytochemical marker for myoepithelial cells (Dempsey et al., '47). We have shown that alkaline phosphatase activity is weaker in the membrane of the epithelial cell when compared to the myoepithelial cell in mouse mammary gland (Russo and Wells, '75b). Since evidence of the presence of this enzyme in cultures of normal human mammary epi-
thelial cells has not been observed, we suggest that the cultures do not contain myoepithelial cells, although i t is possible that culture conditions repress alkaline phosphatase activity. The Mg++-dependent ATPase localized in the plasma membranes of the cells from the later stages in culture suggests that these cells are derived from the duct epithelium of the mammary gland (Russo and Wells, '75a,b). ACKNOWLEDGMENTS
We acknowledge the excellent technical assistance of Clifford Longley and Robert Kaspark, and the assistance of Ms. Ruth Rich and Carolyn Carter in the acquisition of biological resources. LITERATURE CITED Brumrucker, C. R., and T. W. Keenan 1975 Membranes of mammary gland. X. Adenosine triphosphate dependent calcium accumulation by Golgi apparatus rich fractions from bovine mammary gland. Exp. Cell Res., 90: 253-260. Brunk, V. 1973a Distribution and shifts of ingested marker particles in residual bodies and other lysosomes. Exp. Cell Res., 79: 15-27. Brunk, V., J. L. E. Ericsson, J. Pontin and B. Westermark 1973b Residual bodies in cultured human glial cells. Exp. Cell Res., 79: 1-14. Dempsey, E. W., M. Brenting and G. B. Wislocki 1947 Observations on the chemical cytology of the mammary gland. Am. J. Anat., 8 1 : 309-342. Devine, C. E., and A. P. Somlyo 1971 Thick filaments in vascular smooth muscle. J. Cell Biol., 49: 636-649. Flaxman, B. A., and D. P. Chopra 1972 Cell cycle of normal and psoriatic epidermis i n nitro. J. Invest. Dermatol., 59: 102-105. Flaxman, B. A., M. A. Lutzner and E. J. Van Scott 1967 Cell maturation and tissue organization in epithelial outgrowths from skin and buccal mucera in nitro. J. Invest. Dermatol., 49: 322-332. Furmanski, P., C. Longley, D. Fouchey, R. Rich and M. A. Rich 1974a Normal human mammary cells in culture; Evidence for oncornavirus-like particles. J. Natl. Cancer Inst., 52: 975-977. 197410 Oncornavirus-like particles i n human mammary cells in culture. Fed. Proc., 33: 753 (abstract). Graham, R. C., and M. J. Karnovsky 1966 The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique. J. Histochem. Cytochem., 14: 291-302. Hirsch, J. G., and M. E. Fedorko 1968 Ultrastructure of human leukocytes after simulta-
NORMAL HUMAN MAMMARY EPITHELIAL CELLS neous fixation with glutaraldehyde and osmium tetroxide and post-fixation in uranyl acetate. J. Cell Biol., 38: 615-627. Huang, C. M., and T. W. Keenan 1972 Adenosine triphosphatase activity of bovine milk fat globule membranes. Comp. Biochem. Physiol., 43b: 277-282. Ishikawa, H., R. Bischoff and H. Holtzer 1969 Formation of arrowhead complexes with heavy meromyosin in a variety of cell types. J. Cell Biol., 43: 312-328. Jimbow, K., and T. B. Fitzpatrick 1975 Changes in distribution pattern of cytoplasmic filaments in human melanocytes during ultravioletmediated melanin pigmentation. J. Cell Biol., 65: 481-488. Kruse, P. F., Jr., and E. Miedema 1965 Production and characterization of multiple layered population of animal cells. J. Cell Biol., 27: 273-280. Linzell, J. L., and M. Peaker 1971 Intracellular concentrations of sodium, potassium and chloride in the lactating mammary gland and their relation to the secretory mechanism. J. Physiol., 216: 683-700. Luft, J . H. 1961 Improvements i n epoxy resin embedding methods. J. Biophys. Biochem. Cytol., 9: 409-414. Mayahara, H., and K. Ogawa 1968 The effect of thickness of specimen on the ultrastructural localization of alkaline phosphatase activity in the rat proximal convoluted tubule. J. Histochem. Cytochem., 16: 721-724. McNutt, N. S., L. A. Culp and P. H. Black 1973 Contact-inhibited revertant cell lines isolated from SV4O-transformed cells. IV. Microfilaments distribution and cell shape in untransformed, and revertant Balb/c 3T3 cells. J. Cell Biol., 56: 412-428. Mollenhauer, D. M. 1964 Plastic embedding mixtures for use in electron microscopy. Stain. Technol., 39: 111-114. Murad, T. M. 1971 A proposed histochemical and electron microscopic classification of human breast cancer according to cell origin. Cancer, 27: 288-299. Pitelka, D. R., S. T. Hamamonto, J. G. Duafala and M. K. Nemanic 1973 Cell contacts in the mouse mammary gland. I. Normal Gland in postnatal development and the secretory cycle. J. Cell Biol., 56: 797-818. Pollard, T. D., E. Shelton, R. R. Weihing and E. D. Korn 1970 Ultrastructural character. ization of F-actin isolated from Acanthamoeba castellanii and identification of cytoplasmic
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filaments as F-actin by reaction with rabbit meromyosin. J . Mol. Biol., 50: 91-97. Reaven, E. P., and S. G. Axline 1973 Subplasmalemmal microfilaments and microtubules in resting and phagocytizing cultivated macrophages. J. Cell Biol., 59: 12-27. Reynolds, E. S. 1963 The use of lead citrate at high pH as a n electron opaque stain i n electron microscopy. J. Cell Biol., 17: 208-212. Russo, J . 1971 Fine Structure of the Leydig cell during postnatal differentiation of the mouse testes. Anat. Rec., 170: 343-356. Russo, J., and P. Furmanski 1974 Differentiation of human brest epithelial cells in culture. J . Cell Biol., 63: 292a. Russo, J., P. Furmanski and M. A. Rich 1975 An ultrastructural study of normal human mammary epithelial cells i n culture. Am. J . Anat., 142: 221-232. Russo, J., and P. Wells 1975a Na+ Kf dependent ATPase as a specific marker for myoepithelial cells: A n ultrastructural study. Thirtythird Annual E. M. S. A. Meeting, pp. 4 5 4 4 5 5 . 1975b Light microscopic localization of cytochemical reactions i n epoxy-embedded material for electron microscopy. J. Histochem., Cytochem., 23: 921-931. Sanford, K. K., B. E. Barker, M. W. Woods, R. Parshad and L. W. Law 1967 Search for “indicators” of neoplastic conversion in uitro. J . Nat. Cancer Inst., 39: 705-733. Sanford, K. K., T. B. J h n n , B. B. Westfall, A. B. Lovalesky, L. T. Dupree and W. R. Earb 1961 Sarcomatous change and maintainance of differentiation in long term cultures of mouse mammary carcinoma. J. Nat. Cancer Inst., 26: 1139-1 183. Tannenbaum, M., M. Weiss and A. J. Marx 1969 Ultrastructure of the human mammary ductule. Cancer, 23: 958-978. Vreeswijk, J. H. A., J. J. H. H. M. Depont and S. L. Bonting 1975 Absence of ( N a + , K + ) ATPase involvement in lactose production by lactating guinea pig mammary gland. Biochem. Biophys. Acta, 392: 12-19. Wachstein, M., and E. Meisel 1957 A comparative study of enzymatic-staining reactions in the rat kidney with necrobiosis induced by ischaemia and nephrotoxic agents. J. Histochem. Cytochem., 5: 204. Wellings, S. R. 1969 Ultrastructural basis of lactogenesis. In: Lactogenesis, The Initiation of Milk Secretion and Parturition. M. Reynolds S. J. Foley, eds. University of Pennsylvania Press, Philadelphia, Pennsylvania, pp. 5-25.
PLATE 1 E X P L A N A T I O N OF F I G U R E S
1
A post-weaning fluid cell treated to demonstrate alkaline phosphatase. Note that the plasma membrane does not show a reaction product. x 4,800.
2
A post-weaning fluid cell treated to demonstrate peroxidase. Dense bodies (DB ) with peroxidase activity are scattered throughout the cytoplasm. x 3,200.
3 A single cell from a 10-day culture treated to demonstrate peroxidase. Dense bodies (DB) show activity. Mitochondria ( M ) , Golgi apparatus ( G ) and a palisade of filaments and granular material ( P ) . x 5,700. 4
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A portion of the cytoplasm of a single cell treated to demonstrate peroxidase. Dense bodies (DB) are positive. Golgi apparatus ( G ) , mitochondria (M),and a portion of the nucleus are seen. x 14,200.
NORMAL HUMAN MAMMARY EPITHELIAL CELLS Russo, Furmanski, Bradley, Wells and Rich
PLATE 1
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PLATE 2 EXPLANATION O F FIGURES
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5
A single cell from a 10-day culture with numerous lipid droplets ( L ) , short microvilli ( M V ) and rough endoplasmic reticulum (RER). X 5,500.
6
Higher magnification of an area from a single cell, ten days in culture, showing a large number of lipid droplets (L), several rnultivesicular bodies (MVB), and a portion of the nucleus (N). x 13,500.
7
A single cell from a 10-day culture with a well developed Golgi apapparatus (G), numerous lipid droplets ( L ) , mitochondria ( M ) , and rough endoplasmic reticulum (RER). A portion of the nucleus ( N ) is also visible. x 5,500.
8
Cytoplasm of a cell, ten days in culture, demonstrating the formation of lipolysosomes (LS). Lipid droplets ( L ) , the Golgi apparatus (G) and secretory granules (SG) associated with cisternae of the Golgi are also visible. x 20,000.
NORMAL HUMAN MAMMARY EPITHELIAL CELLS Russo, Furmanski, Bradley, Wells and Rich
PLATE 2
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PLATE 3 EXPLANATION OF FIGURES
9
A post-weaning fluid cell treated to demonstrate Mg+ +-dependent ATPase. Note the absence of a reaction product in the plasma membrane. x 3,500.
10
Single cells from a 14-day culture forming cell contacts. This material was treated to demonstrate Mg+ +-dependent ATPase. Arrows indicate sites of positive reaction in the microvilli of the interdigitations between the cells. x 2,700.
11
A stratified layer from a 24-day culture, treated to demonstrate localization of Mg+ +-dependent ATPase. The reaction product appears i n the plasma membranes where the cells are i n contact with one another, as indicated by the arrows. x 4,000.
12
Higher magnification of the area between two single cells from a 14-day culture, similar to the cells shown in figure 10. The Mg++dependent ATPase occurs in the plasma membranes of the microvilli as shown by the arrows. x7,500.
13 A n area between two cells in a stratified layer, similar to that shown in figure 11. The reaction product of the Mg++-dependent ATPase is localized in the plasma membranes of the interdigitations between the cells. x 8,000.
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NORMAL HUMAN MAMMARY EPITHELIAL CELLS Russo, Furmanski, Bradley, Wells and Rich
PLATE 3
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PLATE 4 EXPLANATION OF FIGURES
14 A cluster from a 16-day culture exhibits a multilayered structure with desmosomes ( D ) joining the cells. A number of short microvilli ( M V ) are seen in the cell surfaces. x 5,000. 15
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A stratified layer from a 24-day culture consisting of two types of cells. Desmosomes ( D ) are seen between t w o of the cells in the apical portion, while the cell in the basal portion possesses characteristics that are similar to the single cells seen in a 10-day culture. The cytoplasm of this cell contains numerous lipid droplets ( L ) and several dense bodies (DB). Rough endoplasmic reticulum (RER) is visible in the cytoplasm of the apical cell. Portions of the nuclei ( N ) of the t w o cells are also visible. x 5,700.
NORMAL HUMAN MAMMARY EPITHELIAL CELLS Russo, Furmanski, Bradley, Wells and Rich
PLATE 4
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PLATE 5 EXPLANATION O F FIGURES
16
Low magnification of a stratified layer from a 24-day culture, consisting of four layers of cells. Numerous desmosomes ( D ) are observed between individual cells. Parallel arrays of intermediate filaments are seen adjacent to the nucleus ( N ) i n the apical cell. X 4,400.
17
A junction between two of the stratified layer cells showing desmosomes with long arrays of tonofilaments (TF) which extend into the cytoplasm of the cell. x 33,000.
18 High magnification micrograph showing the ultrastructure of a desmosome from the stratifie3 layer. x 50,000.
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NORMAL HUMAN MAMMARY EPITHELIAL CELLS Russo, Furmanski, Bradley, Wells and Rich
PLATE 5
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PLATE 6 EXPLANATION O F FIGURES
19 A low magnification field of a stratified layer from a 24-day culture showing a number of desmosomes ( D ) between the cells. Short microvilli ( M V ) may be seen in the apical surface of the stratified layer. Cytoplasmic filaments ( F ) , numerous mitochondria and portions of two nuclei ( N ) are also shown. x 5,300.
74
20
A portion of the cytoplasm from a stratified layer cell whose basal surface ( B S ) is in contact with the culture vessel. Dense aggregates of thin filaments, 5-7 m s in diameter, occur adjacent to the plasma membrane at the basal surface. The area within the box corresponds to figure 21. x 43,000.
21
Higher magnification of the thin filaments (TnF), 5-7 mp in diameter, showing both longitundinal sections, indicated by TnF, and cross sections of individual filaments as indicated by a n arrow. x 86,000.
22
A n area of the cytoplasm from a stratified layer cell which shows a bundle of intermediate filaments, 8-10 me in diameter, cut in cross section. x 86,000.
23
A bundle of intermediate filaments (IF), 8-10 mp in diameter, from the cytoplasm of a stratified layer cell. The majority of filaments have been cut in longitudinal section. x 86,000.
24
A portion of the cytoplasm of a stratified layer cell showing thick filaments (TkF), 14-15 m s i n diameter, which are arranged in numerous bundles. Rough endoplasmic reticulum (RER) may be seen in the cytoplasm between these bundles. X 30,000.
25
Higher magnification of the thick filaments (TkF), which are 14-15 m , ~i n diameter. Longitudinal sections of these thick filaments are indicated by TkF, while a cross section of an individual filament is indicated by a n arrow. X 86,000.
NORMAL HUMAN MAMMARY EPITHELIAL CELLS Russo, Furmanski, Bradley, Wells and Rich
PLATE 6
75
PLATE 7 EXPLANATION O F FIGURES
76
26
A stratified layer from a 28-day culture, with a n enormous residual body (RB) in the lower portion of the cell, and several osmiophilic dense bodies (DB). Desmosomes (D), mitochondria, and a number of short microvilli (MV) are also shown. X 5,500.
27
A czystal-like inclusion in a lysosome in the cytoplasm of a stratified layer cell, 28 days in culture. x 33,600.
28
A n area of the cytoplasm of a cell 28 days in culture, showing a large membrane-bounded multivesicular body (MVB ) with a similar body above it. A portion of the nucleus ( N ) is shown in the lower right corner of the micrograph. x 16,600.
29
Higher magnification of an area from a cell 28 days in culture, which shows a large residual body that appears to contain membrane-like material. A portion of the nucleus ( N ) is also present. X 20,000.
NORMAL HUMAN MAMMARY EPITHELIAL CELLS Russo, Furmanski, Bradley, Wells and Rich
PLATE 7
ABSTRACT An ultrastructural and cytochemical study of normal human mammary epithelial cells cultured from post-weaning breast fluids is described. Cells were examined at the time of plating and at intervals up to 28 days in culture. Three different stages in the morphological differentiation of these cells in vitro were observed: (1) the first stage was the formation of a monolayer of single cells, which occurred between days 1 and 10 in culture. The cells in this stage were not interconnected by junctional complexes and lacked Mg++dependent ATPase activity in the plasma membranes, but did contain a large quantity of lipid and exhibited some secretory characteristics. ( 2 ) The second stage, occurring at 10 to 16 days in culture, was characterized by the formation of junctional complexes, the appearance of Mg++-dependent ATPase in the plasma membrane and a decrease in the number of dense bodies with peroxidase activity. ( 3 ) The third stage, occurring at 16 to 28 days in culture, was characterized by the formation of stratified layers of epithelial cells, which were interconnected by a large number of desmosomes with numerous pleomorphic microfilaments. The Mg++-dependentATPase activity in the plasma membrane was retained and the dense bodies with peroxidase activity were rarely observed at this stage. During the last seven days in culture, secondary lysosomes, autophagosomes and residual bodies were prominent in the cells of the stratified layer. After 28 days in culture, the cells began to round up and slough off the culture plate.
We have previously reported that epithelial cells can be isolated from postweaning breast fluids of normal human donors (Furmanski et al., '74a,b). In the presence of autologous serum, these cells will grow in culture with a generation time of 60-72 hours. Morphological studies suggest that these cells are derived from the mammary epithelium, shed into the fluid as a consequence of the normal process of involution following the termination of active lactation (Russo et al., '75). We have observed that these cultures of normal human mammary epithelial cells progress through a distinct series of morphological changes. These changes, which suggest a pattern of cell association and differentiation of mammary epithelium in vitro, are detailed here. MATERIALS AND METHODS
Post-weaning breast fluids obtained from AM. J. ANAT., 145: 57-78.
normal human donors were centrifuged at low speed (200 x g ) . The resulting pellet of cells was resuspended in culture medium and the number of cells determined by hemacytometer count. The culture medium consisted of Dulbecco's modified Eagle's medium containing autologous serum ( 15% v/v), insulin ( 10 pg/ml) and antibiotics, as described previously (Furmanski et al., '74a,b). The cells attached to the bottom of the culture chamber and grew with a doubling time of 6072 hours. Cells from 6, 10, 14, 16, 24 and 28 days after plating were used in this study. Cells isolated from the post-weaning fluids were also examined prior to the initial plating. Accepted October 7, '75. 1 This work was supported by Contract NOI-CP33347 and Grant CA 14100 from the National Cancer Institute and an institutional grant to the Michigan Cancer Foundation from the United Foundation of Detroit, Michigan.
57
58
RUSSO, FURMANSKI, BRADLEY, WELLS AND RICH
The cultures were fixed in 1.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2, for one hour at 4°C. After this initial fixation, the plate was divided into four portions while immersed in cold buffer. One portion of each plate was fixed for two hours at 25°C according to the method of Hirsch and Fedorko ('68) for morphological studies. After fixation, the material was washed in 0.85% NaC1, treated with uranyl acetate, and dehydrated through graded concentrations of ethanol. The monolayers of cells were removed from the plastic plate with propylene oxide, and infiltrated and embedded using Luft's Epon ('61) in a ratio of 1 part mixture A to 1 part mixture B. The other portions of the plate were processed for the cytochemical localization of Mg++dependent ATPase, peroxidase, and alkaline phosphatase. These pieces of the culture plate, which had been fixed for one hour before the plate was divided, were transferred to fresh fixative containing 1.5% glutaraldehyde in 0.1 M cacodylate with 1% sucrose, pH 7.2, and fixed for an additional hour at 4°C. After fixation, the portions of the plate were washed in 0.1 M cacodylate with 6% sucrose at 4°C.
A small sample of cells isolated directly from post-weaning fluids was removed prior to the initial plating and fixed by suspension in 1.5% glutaraldehyde for two hours at 4°C. After fixation, the cells were pelleted in a Beckman Microfuge and washed by resuspension in cold buffer. All cytochemical material was held at 4°C in the last buffer wash until the appropriate substrate mixtures were added. Enzyme substrates were used immediately after preparation. The Mg++-dependent ATPase cytochemical reaction was performed using the incubation mixture described by Wachstein and Meisel ('57) and incubated at 37°C for 20 minutes. The cytochemical localization of peroxidase was accomplished using the incubation medium described by Graham and Karnovsky ('66) and incubated for 20 minutes at room temperature. The alkaline phosphatase reaction was performed using the method of Mayahara and Ogawa ('68), and incubated at 37°C for 60 minutes. Material was incubated simultaneously
using control media for each of these enzyme reactions. Control media were prepared by replacing the appropriate cytochemical substrate with an equal volume of distilled water. After incubation, the reaction mixtures were drawn off and the cells or monolayers were washed in 0.1 M cacodylate with 6 % sucrose at 4°C. The cells were postfixed in 1% osmium tetroxide in 0.1 M cacodylate for two hours, washed with buffer and dehydrated using ethanol. The monolayer of cells was removed from the plastic plate with propylene oxide. All material for cytochemical study was infiltrated and embedded using an EponAraldite mixture (Mollenhauer, '64). Thin sections were cut for electron microscopy using a diamond knife mounted in an LKB Ultrotome and examined with either a Siemens Elmiskop 1A at 60 kv or with a Hitachi HU-1lA at 75 kv. The majority of sections that were observed and photographed were stained with 2% uranyl acetate and Reynold's lead citrate ('63). However, some sections from the cytochemical blocks were stained only with 2% uranyl acetate in order to evaluate the histochemical localizations without the effect of lead staining. RESULTS
In cultures of normal human mammary epithelial cells obtained from post-weaning fluids, three distinct stages of morphological differentiation of the cells were observed during the 28-day culture period. The first stage, which occurred during the first ten days in culture, consisted of a monolayer of single cells in which no junctional complexes were observed. The second stage, observed during the tenth to sixteenth day, consisted of clusters of four to eight epithelial cells which were connected by desmosomes and junctional complexes. The third stage, from 16 to 28 days, was characterized by stratified layers of epithelial cells with large numbers of desmosomes and tight junctions interconnecting the cells. Cells f r o m post-weaning fluid The epithelial cells obtained directly from post-weaning fluids possessed a high concentration of lipid droplets, with few
NORMAL HUMAN MAMMARY EPITHELIAL CELLS
mitochondria, some secretory granules, and a poorly developed endoplasmic reticulum (figs. 1, 2, 9). The cell surface had numerous microvilli of varying size and shape, The nuclei of these cells had a regular shape with some indentations, typical of the nuclei of normal mammary epithelium (Tannebaum et al., '69). One or two nucleoli were usually present in each nucleus (figs. 1, 2 ) . The cells obtained directly from postweaning fluids did not show any reaction product in their plasma membranes for Mg++-dependentATPase (fig. 9 ) or alkaline phosphatase (fig. 1 ) . However, they did show a small number of bodies with peroxidase activity (fig. 2 ) .
The monolayer During the first ten days in culture, two cellular types were observed in the monolayer. The first was a flat cell (fig. 3 ) with a small number of elongated microvilli and few lipid droplets. The Golgi complex was located in a supra-nuclear position. The nuclei were generally ovoid i n shape and located in the basal portion of the cells. Heterochromatic material was scarce and was attached to the inner membrane of the nuclear envelope. The second type of cell was more spherical or elevated in nature (figs. 5, 7 ) . These cells had a larger number of microvilli on their surface and possessed a higher concentration of lipid droplets i n their cytoplasm. The Golgi complex was well developed and usually found in the apical portion between the free border of the cell and the nucleus, which resembles the polarity of glandular epithelium (fig. 7). Some granules containing electron dense material were observed in proximity to the Golgi complex (fig. 8 ) . Multivesicular bodies were observed frequently at this stage (fig. 6 ) and lipid droplets were often seen apparently fusing with lysosomes to form lipolysosomes (fig. 8). Rough endoplasmic reticulum was present (fig. 5 ) and free ribosomes were dispersed throughout the cytoplasm. The nuclei of these cells were relatively spherical and were frequently observed in the basal portion, but rarely in the apical portion of these cells (fig. 7 ) . Nucleoli were found either in the
59
center of the nucleus or associated with the nuclear envelope (figs. 5, 7). Both types of cells became firmly attached to the petri dish and a palisade of thin microfilaments 5-7 mp in diameter (figs. 20, 21) and granular material was found adjacent to the attached surface of these cells (fig. 3 ) . Although the cells in the monolayer were connected by interdigitations (fig. 5), typical junctional complexes were not observed. The plasma membranes of these cells did not exhibit Mg' +-dependent ATPase activity. Toward the end of this stage, the cells in the monolayer came into contact with each other and began to form multicellular clusters (fig. 10). Mg'+-dependent ATPase activity was detected in the plasma membranes of the interdigitations that occurred between these cells (figs. 10, 12). The flat cells of the monolayer possessed a number of dense bodies with peroxidase activity as demonstrated by incubation with 3,3' diaminobenzidine (figs. 3, 4 ) . The plasma membranes of the monolayer cells were negative for alkaline phosphatase activity, a s were the plasma membranes of the cells obtained directly from post-weaning fluids.
Multicellulnr clusters Between 10 and 16 days in culture, the confluent monolayers were characterized by well developed junctional complexes (Russo et al., '75). Scattered among the monolayers were clusters of four to ten cells that were joined together by numerous desmosomes (fig. 14). Cell surfaces showed a number of short and blunt microvilli. Mitochondria were scattered throughout the cytoplasm and were polymorphic with many transverse cristae. The rough endoplasmic reticulum was poorly developed but was more abundant than the smooth endoplasmic reticulum. Few inclusions and a decreased number of lysosomes were observed i n these cells. A distinct feature was the appearance of tonofilaments which were associated with the terminal web of the desmosomes (figs. 17, 18). The nuclei of the cells were found in the apical portion and were polymorphic, with numerous indentations. Scarce heterochromatic material and usually one nucleolus were seen (fig. 14). Nuclei in the
60
RUSSO, FURMANSKI, BRADLEY, WELLS AND RICH
basal portion of the cluster were more elongated with an even distribution of heterochromatic material. Dense bodies, positive for the 3,3' diaminobenzidine reaction, were absent. Mg++-dependent ATPase was present on the cell surface and between the cells. The alkaline phosphatase cytochemical reaction was negative, as in the preceding stages. Stratified layers The formation of clusters was followed by the growth of cells in stratified layers, occurring generally after 16 days in culture and persisting until the cells rounded up and sloughed off the culture plate. The stratification consisted of two to six layers of cells joined together by numerous desmosomes (figs. 15, 16). These cells were flatter than those observed in the clusters described above. In some of these structures, cells with morphological characteristics very similar to those observed during the first week in culture were observed in the basal strata (fig. 15). These cells exhibited numerous lipid droplets and dense bodies with peroxidase activity, suggesting that they had originated from the monolayer cells by cell division and morphological differentiation. Only a few short and blunt microvilli were observed at the cell surfaces (figs. 17, 19, 26). The most prominent feature of the cytoplasm was the presence of numerous bundles of microfilaments (figs. 16, 19, 24). These filaments may be classified according to their diameter and distribution into three different types. The first type, 5-7 mp in diameter, was usually found in the basal portion of the cell adjacent to the surface that was attached to the culture vessel (figs. 20, 21). The second type of filament was found in bundles scattered throughout the cytoplasm (fig. 24) and these thick filaments were 14-15 mp in diameter (fig. 25). The third type was intermediate in diameter, 8-10 mp, and these filaments were observed in parallel arrays in the cytoplasm (figs. 22, 23) and adjacent to the nucleus (fig. 16). Tonofibrils were always found associated with the terminal web of the desmosomes (figs. 16-19). They appeared in long arrays originating from the desmosomes and extending into the cytoplasm.
Their diameters ranged from 7-10 mp and they were similar in size and appearance to the intermediate filaments described above. Numerous polymorphic mitochondria were scattered throughout the cytoplasm (figs. 19, 26). The Golgi complex was no longer visible in these cells. The rough endoplasmic reticulum was poorly developed (figs. 15, 24), and free ribosomes were scattered throughout the cytoplasm (figs. 17, 19). The nuclei were polymorphic and heterochromatic material was scarce or absent. Usually only one nucleolus was present (figs. 15, 16, 19, 26). Between 24 and 28 days in culture, several types of lysosomes appeared in the cells of the stratified layers. Secondary lysosomes, autophagosomes, and residual bodies became a prominent element and occupied a major portion of the cytoplasm (figs. 26, 28, 29). The multivesicular bodies (fig. 28) could be classified as secondary or autophagic lysosomes. The degenerative bodies or residual bodies were considered to be tertiary lysosomes in which indigestible cellular debris was concentrated during the final stages in culture (figs. 26, 29). Whorl-like or myelinlike structures were observed within the residual or degenerative bodies (figs. 26, 29). Crystal-like structures were often seen associated with lysosomes in the cytoplasm of these cells (fig. 27). During the final stages of culture, a strong Mg++-dependentATPase cytochemical reaction was observed on membranes between the cells (figs. 11, 13). However, in contrast to the monolayer cells previously described, the bodies with peroxidase activity were only rarely observed. Following four weeks in culture, the cells rounded up and sloughed off the surface of the culture vessel. DISCUSSION
Post-weaning fluids from normal human donors contain epithelial cells that can be grown in culture (Furmanski et al., '74a,b; Russo et al., '75). When these cells are plated, they attach to the bottom of the dish and grow to form confluent monolayers. One cell type observed in these cultures is seen only during the first ten days and
NORMAL HUMAN MAMMARY EPITHELIAL CELLS
in low concentrations. The large number of dense bodies with peroxidase activity and the elongated microvilli suggest that these cells are macrophages. Similar characteristics have been described for monocytes and macrophages in culture (Reaven and Axline, '73). Another type of cell observed during the first ten days in culture exhibits many features characteristic of breast epithelium, as previously described (Russo et al., '75). These cells possess a marked polarity demonstrated by microvilli on the free surface and the supranuclear position of the Golgi apparatus, which was associated with electron dense granules that resembled secretory products. The well developed Golgi apparatus, the rough endoplasmic reticulum and the increased quantity of lipid droplets which are present in these cells and in the post-weaning fluid cells have also been observed in lactating mammary epithelium (Wellings, '68). However, possibly due to the absence of hormones which are normaly present in the human breast during lactation, these cells in culture do not appear to release secretory products. Our observations suggest that the digestion of lipid droplets and the formation of lipolysosomes occur during this phase in culture, and that this process leads to the reduction of lipid that is observed in the later stages. Lipolysosomes have also been demonstrated to play a role in the process of cellular differentiation of the Leydig cells in the testes of the mouse (Russo, '71). During the second stage, from 10 to 16 days in culture, multilayered clusters of cells are formed which are the morphological precursors of the stratified layer. Most of the characteristics which were described for the monolayer are not observed in the clusters; the Golgi complex is diminished and the lipid droplets are much scarcer, Clusters of cells may be formed in a manner suggested by figure 10, in which the microvilli of these cells have come in contact and the cells are beginning to grow on top of one another. When the cells begin to join one another, Mg++dependent ATPase activity appears in the plasma membrane and, later, desmosomes and permanent junctions are formed.
61
At the present time, the function of ATPase activity in the plasma membranes of lactating mammary epithelium is not completely understood. Localizations of ATPase activity in lactating mammary gland are not in agreement with biochemical and physiological data (Huang and Keenan, '72, Baumrucker and Keenan, '75, Linzell and Peaker, '71). Other authors have established that this enzyme is not directly involved in the process of lactose secretion (Vreeswijk, '75). However, we have demonstrated that Mg' +-activated ATPase is a permanent marker for epithelial cells in the resting mammary gland (Russo and Wells, '75a,b). Similar results have been reported in pathological conditions of the breast (Murad, '71). In mouse mammary gland, Pitelka ('73) observed that the desmosomes which are present in the resting gland disappear, and only tight and gap junctions remain during lactation. The lack of desmosomes and ATPase activity, in addition to the morphology of the post-weaning fluid cells and the first-stage cells in culture, suggest that these cells are in a secretory state. The correlation between the appearance of desmosomes and the detection of ATPase activity in the plasma membrane after ten days in culture suggests a transition from a secretory state to a resting state. The formation of stratified layers is one of the characteristics of these human breast cells in culture. Unlike those described by Kruse and Miedema ('65), these multilayered structures do not suggest characteristics of transformation in vitro but appear to reflect the cell type and the conditions of culture (Sanford, '61, '67). A characteristic of normal diploid cells is finite growth in culture. The capacity of normal cells to exhibit aging and senescence is a n important marker in differentiating normal from transformed cells. One of the criteria for aging is the presence of residual bodies, autophagolysosomes, and lipofucsin granules. The increase of these organelles with age has been described by Brunk ('73a,b). The accumulation of residual bodies, described as an indication of the finite lifespan of normal cells in culture (Brunk, '73a), was observed during the fourth week, just prior
62
RUSSO, FURMANSKI, BRADLEY, WELLS AND RICH
to the expiration of the cultures described in this report. The process of cellular degeneration, which occurs in these breast epithelial cells, differs from the processes of cellular degeneration and keratinization which occur in the epithelium of the epidermis (Flaxman et al., '67). The cytoplasmic filaments which were observed in the stratified layer cells have been grouped according to their diameter and morphology. The first class of filaments, which are 5-7 mp in diameter, has been observed in other types of cells in vitro and in vivo and has been shown to possess actin-like properties (Ishikawa et al., '69; Pollard et al., '70). The second class of filaments, with diameters of 1415 mp, has an appearance similar to the thick myofilaments which have been observed in smooth muscle cells (Devine and Somlyo, '71). The third class of filaments, with diameters of 8-10 mp, is oriented in bundles in the cytoplasm. Similar arrangements of filaments of this diameter have been found in fibroblasts and melanocytes (McNutt et al., '73; Jimbow and Fitzpatrick, '75). A massive accumulation of filaments in the cytoplasm has been related to the process of keratinization that is observed in the epithelium of the skin. This keratinization is also followed by other changes such as nuclear degeneration, the disappearance of ribosomes and mitochondria, and alterations of the desmosomes (Flaxman et al., '67, '72). These changes in cellular organelles were not observed in the human breast epithelial cells in culture, and evidence of keratohyaline or precursors of keratin were not detectable. Histochemical assays for plasma membrane alkaline phosphatase were negative in both the post-weaning fluid cells and the cells in all stages during culture. In the mammary gland, alkaline phosphatase has been shown to be a cytochemical marker for myoepithelial cells (Dempsey et al., '47). We have shown that alkaline phosphatase activity is weaker in the membrane of the epithelial cell when compared to the myoepithelial cell in mouse mammary gland (Russo and Wells, '75b). Since evidence of the presence of this enzyme in cultures of normal human mammary epi-
thelial cells has not been observed, we suggest that the cultures do not contain myoepithelial cells, although i t is possible that culture conditions repress alkaline phosphatase activity. The Mg++-dependent ATPase localized in the plasma membranes of the cells from the later stages in culture suggests that these cells are derived from the duct epithelium of the mammary gland (Russo and Wells, '75a,b). ACKNOWLEDGMENTS
We acknowledge the excellent technical assistance of Clifford Longley and Robert Kaspark, and the assistance of Ms. Ruth Rich and Carolyn Carter in the acquisition of biological resources. LITERATURE CITED Brumrucker, C. R., and T. W. Keenan 1975 Membranes of mammary gland. X. Adenosine triphosphate dependent calcium accumulation by Golgi apparatus rich fractions from bovine mammary gland. Exp. Cell Res., 90: 253-260. Brunk, V. 1973a Distribution and shifts of ingested marker particles in residual bodies and other lysosomes. Exp. Cell Res., 79: 15-27. Brunk, V., J. L. E. Ericsson, J. Pontin and B. Westermark 1973b Residual bodies in cultured human glial cells. Exp. Cell Res., 79: 1-14. Dempsey, E. W., M. Brenting and G. B. Wislocki 1947 Observations on the chemical cytology of the mammary gland. Am. J. Anat., 8 1 : 309-342. Devine, C. E., and A. P. Somlyo 1971 Thick filaments in vascular smooth muscle. J. Cell Biol., 49: 636-649. Flaxman, B. A., and D. P. Chopra 1972 Cell cycle of normal and psoriatic epidermis i n nitro. J. Invest. Dermatol., 59: 102-105. Flaxman, B. A., M. A. Lutzner and E. J. Van Scott 1967 Cell maturation and tissue organization in epithelial outgrowths from skin and buccal mucera in nitro. J. Invest. Dermatol., 49: 322-332. Furmanski, P., C. Longley, D. Fouchey, R. Rich and M. A. Rich 1974a Normal human mammary cells in culture; Evidence for oncornavirus-like particles. J. Natl. Cancer Inst., 52: 975-977. 197410 Oncornavirus-like particles i n human mammary cells in culture. Fed. Proc., 33: 753 (abstract). Graham, R. C., and M. J. Karnovsky 1966 The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique. J. Histochem. Cytochem., 14: 291-302. Hirsch, J. G., and M. E. Fedorko 1968 Ultrastructure of human leukocytes after simulta-
NORMAL HUMAN MAMMARY EPITHELIAL CELLS neous fixation with glutaraldehyde and osmium tetroxide and post-fixation in uranyl acetate. J. Cell Biol., 38: 615-627. Huang, C. M., and T. W. Keenan 1972 Adenosine triphosphatase activity of bovine milk fat globule membranes. Comp. Biochem. Physiol., 43b: 277-282. Ishikawa, H., R. Bischoff and H. Holtzer 1969 Formation of arrowhead complexes with heavy meromyosin in a variety of cell types. J. Cell Biol., 43: 312-328. Jimbow, K., and T. B. Fitzpatrick 1975 Changes in distribution pattern of cytoplasmic filaments in human melanocytes during ultravioletmediated melanin pigmentation. J. Cell Biol., 65: 481-488. Kruse, P. F., Jr., and E. Miedema 1965 Production and characterization of multiple layered population of animal cells. J. Cell Biol., 27: 273-280. Linzell, J. L., and M. Peaker 1971 Intracellular concentrations of sodium, potassium and chloride in the lactating mammary gland and their relation to the secretory mechanism. J. Physiol., 216: 683-700. Luft, J . H. 1961 Improvements i n epoxy resin embedding methods. J. Biophys. Biochem. Cytol., 9: 409-414. Mayahara, H., and K. Ogawa 1968 The effect of thickness of specimen on the ultrastructural localization of alkaline phosphatase activity in the rat proximal convoluted tubule. J. Histochem. Cytochem., 16: 721-724. McNutt, N. S., L. A. Culp and P. H. Black 1973 Contact-inhibited revertant cell lines isolated from SV4O-transformed cells. IV. Microfilaments distribution and cell shape in untransformed, and revertant Balb/c 3T3 cells. J. Cell Biol., 56: 412-428. Mollenhauer, D. M. 1964 Plastic embedding mixtures for use in electron microscopy. Stain. Technol., 39: 111-114. Murad, T. M. 1971 A proposed histochemical and electron microscopic classification of human breast cancer according to cell origin. Cancer, 27: 288-299. Pitelka, D. R., S. T. Hamamonto, J. G. Duafala and M. K. Nemanic 1973 Cell contacts in the mouse mammary gland. I. Normal Gland in postnatal development and the secretory cycle. J. Cell Biol., 56: 797-818. Pollard, T. D., E. Shelton, R. R. Weihing and E. D. Korn 1970 Ultrastructural character. ization of F-actin isolated from Acanthamoeba castellanii and identification of cytoplasmic
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filaments as F-actin by reaction with rabbit meromyosin. J . Mol. Biol., 50: 91-97. Reaven, E. P., and S. G. Axline 1973 Subplasmalemmal microfilaments and microtubules in resting and phagocytizing cultivated macrophages. J. Cell Biol., 59: 12-27. Reynolds, E. S. 1963 The use of lead citrate at high pH as a n electron opaque stain i n electron microscopy. J. Cell Biol., 17: 208-212. Russo, J . 1971 Fine Structure of the Leydig cell during postnatal differentiation of the mouse testes. Anat. Rec., 170: 343-356. Russo, J., and P. Furmanski 1974 Differentiation of human brest epithelial cells in culture. J . Cell Biol., 63: 292a. Russo, J., P. Furmanski and M. A. Rich 1975 An ultrastructural study of normal human mammary epithelial cells i n culture. Am. J . Anat., 142: 221-232. Russo, J., and P. Wells 1975a Na+ Kf dependent ATPase as a specific marker for myoepithelial cells: A n ultrastructural study. Thirtythird Annual E. M. S. A. Meeting, pp. 4 5 4 4 5 5 . 1975b Light microscopic localization of cytochemical reactions i n epoxy-embedded material for electron microscopy. J. Histochem., Cytochem., 23: 921-931. Sanford, K. K., B. E. Barker, M. W. Woods, R. Parshad and L. W. Law 1967 Search for “indicators” of neoplastic conversion in uitro. J . Nat. Cancer Inst., 39: 705-733. Sanford, K. K., T. B. J h n n , B. B. Westfall, A. B. Lovalesky, L. T. Dupree and W. R. Earb 1961 Sarcomatous change and maintainance of differentiation in long term cultures of mouse mammary carcinoma. J. Nat. Cancer Inst., 26: 1139-1 183. Tannenbaum, M., M. Weiss and A. J. Marx 1969 Ultrastructure of the human mammary ductule. Cancer, 23: 958-978. Vreeswijk, J. H. A., J. J. H. H. M. Depont and S. L. Bonting 1975 Absence of ( N a + , K + ) ATPase involvement in lactose production by lactating guinea pig mammary gland. Biochem. Biophys. Acta, 392: 12-19. Wachstein, M., and E. Meisel 1957 A comparative study of enzymatic-staining reactions in the rat kidney with necrobiosis induced by ischaemia and nephrotoxic agents. J. Histochem. Cytochem., 5: 204. Wellings, S. R. 1969 Ultrastructural basis of lactogenesis. In: Lactogenesis, The Initiation of Milk Secretion and Parturition. M. Reynolds S. J. Foley, eds. University of Pennsylvania Press, Philadelphia, Pennsylvania, pp. 5-25.
PLATE 1 E X P L A N A T I O N OF F I G U R E S
1
A post-weaning fluid cell treated to demonstrate alkaline phosphatase. Note that the plasma membrane does not show a reaction product. x 4,800.
2
A post-weaning fluid cell treated to demonstrate peroxidase. Dense bodies (DB ) with peroxidase activity are scattered throughout the cytoplasm. x 3,200.
3 A single cell from a 10-day culture treated to demonstrate peroxidase. Dense bodies (DB) show activity. Mitochondria ( M ) , Golgi apparatus ( G ) and a palisade of filaments and granular material ( P ) . x 5,700. 4
64
A portion of the cytoplasm of a single cell treated to demonstrate peroxidase. Dense bodies (DB) are positive. Golgi apparatus ( G ) , mitochondria (M),and a portion of the nucleus are seen. x 14,200.
NORMAL HUMAN MAMMARY EPITHELIAL CELLS Russo, Furmanski, Bradley, Wells and Rich
PLATE 1
65
PLATE 2 EXPLANATION O F FIGURES
66
5
A single cell from a 10-day culture with numerous lipid droplets ( L ) , short microvilli ( M V ) and rough endoplasmic reticulum (RER). X 5,500.
6
Higher magnification of an area from a single cell, ten days in culture, showing a large number of lipid droplets (L), several rnultivesicular bodies (MVB), and a portion of the nucleus (N). x 13,500.
7
A single cell from a 10-day culture with a well developed Golgi apapparatus (G), numerous lipid droplets ( L ) , mitochondria ( M ) , and rough endoplasmic reticulum (RER). A portion of the nucleus ( N ) is also visible. x 5,500.
8
Cytoplasm of a cell, ten days in culture, demonstrating the formation of lipolysosomes (LS). Lipid droplets ( L ) , the Golgi apparatus (G) and secretory granules (SG) associated with cisternae of the Golgi are also visible. x 20,000.
NORMAL HUMAN MAMMARY EPITHELIAL CELLS Russo, Furmanski, Bradley, Wells and Rich
PLATE 2
67
PLATE 3 EXPLANATION OF FIGURES
9
A post-weaning fluid cell treated to demonstrate Mg+ +-dependent ATPase. Note the absence of a reaction product in the plasma membrane. x 3,500.
10
Single cells from a 14-day culture forming cell contacts. This material was treated to demonstrate Mg+ +-dependent ATPase. Arrows indicate sites of positive reaction in the microvilli of the interdigitations between the cells. x 2,700.
11
A stratified layer from a 24-day culture, treated to demonstrate localization of Mg+ +-dependent ATPase. The reaction product appears i n the plasma membranes where the cells are i n contact with one another, as indicated by the arrows. x 4,000.
12
Higher magnification of the area between two single cells from a 14-day culture, similar to the cells shown in figure 10. The Mg++dependent ATPase occurs in the plasma membranes of the microvilli as shown by the arrows. x7,500.
13 A n area between two cells in a stratified layer, similar to that shown in figure 11. The reaction product of the Mg++-dependent ATPase is localized in the plasma membranes of the interdigitations between the cells. x 8,000.
68
NORMAL HUMAN MAMMARY EPITHELIAL CELLS Russo, Furmanski, Bradley, Wells and Rich
PLATE 3
69
PLATE 4 EXPLANATION OF FIGURES
14 A cluster from a 16-day culture exhibits a multilayered structure with desmosomes ( D ) joining the cells. A number of short microvilli ( M V ) are seen in the cell surfaces. x 5,000. 15
70
A stratified layer from a 24-day culture consisting of two types of cells. Desmosomes ( D ) are seen between t w o of the cells in the apical portion, while the cell in the basal portion possesses characteristics that are similar to the single cells seen in a 10-day culture. The cytoplasm of this cell contains numerous lipid droplets ( L ) and several dense bodies (DB). Rough endoplasmic reticulum (RER) is visible in the cytoplasm of the apical cell. Portions of the nuclei ( N ) of the t w o cells are also visible. x 5,700.
NORMAL HUMAN MAMMARY EPITHELIAL CELLS Russo, Furmanski, Bradley, Wells and Rich
PLATE 4
71
PLATE 5 EXPLANATION O F FIGURES
16
Low magnification of a stratified layer from a 24-day culture, consisting of four layers of cells. Numerous desmosomes ( D ) are observed between individual cells. Parallel arrays of intermediate filaments are seen adjacent to the nucleus ( N ) i n the apical cell. X 4,400.
17
A junction between two of the stratified layer cells showing desmosomes with long arrays of tonofilaments (TF) which extend into the cytoplasm of the cell. x 33,000.
18 High magnification micrograph showing the ultrastructure of a desmosome from the stratifie3 layer. x 50,000.
72
NORMAL HUMAN MAMMARY EPITHELIAL CELLS Russo, Furmanski, Bradley, Wells and Rich
PLATE 5
73
PLATE 6 EXPLANATION O F FIGURES
19 A low magnification field of a stratified layer from a 24-day culture showing a number of desmosomes ( D ) between the cells. Short microvilli ( M V ) may be seen in the apical surface of the stratified layer. Cytoplasmic filaments ( F ) , numerous mitochondria and portions of two nuclei ( N ) are also shown. x 5,300.
74
20
A portion of the cytoplasm from a stratified layer cell whose basal surface ( B S ) is in contact with the culture vessel. Dense aggregates of thin filaments, 5-7 m s in diameter, occur adjacent to the plasma membrane at the basal surface. The area within the box corresponds to figure 21. x 43,000.
21
Higher magnification of the thin filaments (TnF), 5-7 mp in diameter, showing both longitundinal sections, indicated by TnF, and cross sections of individual filaments as indicated by a n arrow. x 86,000.
22
A n area of the cytoplasm from a stratified layer cell which shows a bundle of intermediate filaments, 8-10 me in diameter, cut in cross section. x 86,000.
23
A bundle of intermediate filaments (IF), 8-10 mp in diameter, from the cytoplasm of a stratified layer cell. The majority of filaments have been cut in longitudinal section. x 86,000.
24
A portion of the cytoplasm of a stratified layer cell showing thick filaments (TkF), 14-15 m s i n diameter, which are arranged in numerous bundles. Rough endoplasmic reticulum (RER) may be seen in the cytoplasm between these bundles. X 30,000.
25
Higher magnification of the thick filaments (TkF), which are 14-15 m , ~i n diameter. Longitudinal sections of these thick filaments are indicated by TkF, while a cross section of an individual filament is indicated by a n arrow. X 86,000.
NORMAL HUMAN MAMMARY EPITHELIAL CELLS Russo, Furmanski, Bradley, Wells and Rich
PLATE 6
75
PLATE 7 EXPLANATION O F FIGURES
76
26
A stratified layer from a 28-day culture, with a n enormous residual body (RB) in the lower portion of the cell, and several osmiophilic dense bodies (DB). Desmosomes (D), mitochondria, and a number of short microvilli (MV) are also shown. X 5,500.
27
A czystal-like inclusion in a lysosome in the cytoplasm of a stratified layer cell, 28 days in culture. x 33,600.
28
A n area of the cytoplasm of a cell 28 days in culture, showing a large membrane-bounded multivesicular body (MVB ) with a similar body above it. A portion of the nucleus ( N ) is shown in the lower right corner of the micrograph. x 16,600.
29
Higher magnification of an area from a cell 28 days in culture, which shows a large residual body that appears to contain membrane-like material. A portion of the nucleus ( N ) is also present. X 20,000.
NORMAL HUMAN MAMMARY EPITHELIAL CELLS Russo, Furmanski, Bradley, Wells and Rich
PLATE 7
