Intrinsic and Extrinsic Regulation of the ... - Semantic Scholar
Cell, Vol. 22,
17-25.
November
1980
(Part
1). Copyright
0 1960
by MIT
Intrinsic and Extrinsic Regulation of the Differentiation of Skin, Cornea1 and Esophageal Epithelial Cells Thomas
I. Doran,
Alda Vidrich
Departments of Dermatology, and Anatomy The Johns Hopkins University Baltimore, Maryland 21205
and Tung-Tien and
of Cell
School
Sun*
Biology
of Medicine
Summary Basal cells of the stratified squamous epithelia of rabbit skin, cornea and esophagus appear morphologically similar. However, the histological features of their subsequent differentiation are different, and the three epithelia are characterized by distinctive keratin proteins. To analyze the relative importance of intrinsic versus extrinsic factors in regulating the differentiation of these epithelia, we compared their behavior under identical in vitro and in vivo conditions. When cultured in the presence of 3T3 feeder cells, keratinocytes from all three epithelia formed differentiating colonies. Although in culture the three cell types approached a common phenotype, they remained distinguishable morphologically and, in some cases, biochemically. When these cultured epithelial cells were trypsinized, suspended in medium and injected subcutaneously into athymic (nude) mice, each of the three cell types generated a characteristic cyst consisting of stratified squamous epithelium. Cultured skin, cornea1 and esophageal keratinocytes formed epithelia which were keratinized, nonkeratinized and parakeratinized, respectively. In addition, the injected skin and esophageal epithelial cells reacquired their distinctive in vivo keratin patterns. These data suggest that the three epithelia are not equipotential. Furthermore, since the distinctive in vivo phenotype of each epithelium was expressed when the cells were transplanted to the same subcutaneous site, the expression of these differences does not depend on specific mesenchymal instruction but on permissive factors not present in the culture system. Thus under in vivo conditions intrinsic divergence must play a predominant role in determining the characteristic phenotypes of the three epithelia. On the other hand, the finding that the morphological and biochemical differentiation of a given epithelium can be reversibly modulated by the external environment demonstrates that extrinsic factors may, under certain conditions, also play a role in regulating epithelial differentiation.
These multiple layered epithelia are composed mainly of keratinocytes which contain abundant keratin filaments (Sun and Green, 1977, 1978a). Although the basal cells of different stratified squamous epithelia appear morphologically similar, their subsequent differentiation can be very different; hence the descriptions of keratinized epidermis, nonkeratinized cornea1 epithelium and parakeratinized (rabbit) esophageal epithelium (Alvares and Meyer, 1971). Normally the histological features of these various epithelia are faithfully conserved throughout post natal life. To explain such a conservation of tissue specificity, two hypotheses, which are not mutally exclusive, have been proposed. First, despite their apparent similarities, the basal cells of different epithelia may have diverged irreversibly from each other during development (intrinsic divergence), thus resulting in different programs of differentiation. Alternatively, basal cells of different stratified squamous epithelia may be equipotential and therefore identical to each other (Billingham and Silvers, 1963). Their distinctive in vivo differentiation would then be due to different local, directive influences derived from their environment, for example, from the underlying mesenchymal tissues (external modulation; for reviews of studies using a transplantation approach, see Billingham and Silvers, 1963, 1967; Wessells, 1967). In the presence of lethally irradiated 3T3 feeder cells, epidermal cells undergo clonal growth and retain several important in vivo differentiation markers (Rheinwald and Green, 1975; Sun and Green, 1976, 1978a, 1978b). However, their differentiation is not identical to that of the intact epidermis, since neither keratohyalin granules nor an enucleated stratum corneum are formed. Furthermore, cultured epidermal cells lack the largest keratin proteins (63K-68K daltons) found in the epidermis in vivo (Fuchs and Green, 1978, 1980; Steinert and Yuspa, 1978; Sun and Green, 1978b). In this paper we show that cultured epidermal cells regained their in vivo phenotypes when placed subcutaneously in athymic (nude) mice. The same subcutaneous environment also allowed cultured cornea1 and esophageal epithelial cells to express their distinctive in vivo patterns of differentiation. These results suggest that the three epithelia in (juvenile) animals are intrinsically different from each other and represent different cell lineages.
Results
Introduction
In Vitro
Differentiation
Stratified cornea,
Cultured lial cells fibroblast squamous proteins
rabbit skin, cornea1 and esophageal epithebehaved similarly: all three required suitable support for clonal growth, formed stratified structures and synthesized similar keratin (see below). However, consistent with pre-
squamous esophagus
epithelia line and several
the surfaces of skin, other body sites.
l TO whom reprint requests should be addressed Johns Hopkins Medical School, Baltimore, Maryland
at: Blalock 21205.
926,
of Rabbit
Epithelial
Cells
Cell 18
vious findings (Sun and Green, 1977). the three cultured rabbit cell types remained distinguishable from each other by several criteria. Minor but reproducible morphological differences could be discerned, particularly in colonies of fewer than -lo3 cells (Figures 1 a-l c). Cornified envelopes, a terminal differentiation product of some keratinocytes (Sun and Green, 1976), were formed by surface-cultured rabbit skin and esophageal epithelial cells, but not by cornea1 epithelial cells (compare Sun and Green, 1977). In addition, cornea1 cells elaborated two proteins (64K and 41K) not present in either epidermal (Sun and Green, 1977) or esophageal cells (see below). To examine whether such cultured epithelial cells retained their ability to differentiate normally in an in vivo environment, we studied their behavior in athymic mice, which are immunodeficient and thus can tolerate heterotransplants (Fogh and Giovanella, 1978). Morphological Differentiation of Cultured Epidermal Cells in Athymic Mice Primary cultures (1 O-l 6 days old) of rabbit epidermal cells were trypsinized and aliquots containing 5 x 1 O6 suspended cells were injected subcutaneously into the flanks of athymic mice (Figure 2b). Within two days these cells formed cysts (2-4 mm in diameter) with an epithelial structure lining the cavity and some cell debris in the center. Keratohyalin granules, previously absent in culture, appeared in the cystic epithelium (Figure 2~). The epithelium in day 3 cysts (Figure 2d) became similar to the keratinized epidermis in vivo; it had a well organized stratified squamous structure, prominent keratohyalin granules and an enucleated stratum corneum (Figure 2d). After day 3 the histological features of the cystic epithelium remained essentially unchanged, except for an increase in the number of stratum corneum layers and a decrease in that of living layers (Figure 3e). A similar formation of keratinized cysts (in shortterm experiments of up to 5 days) was observed when cultured epidermal cells were injected subcutaneously into normal BALB/c instead of athymic mice. Thus the results described were not due to conditions peculiar to the athymic nature of the recipient mice. To determine whether cells multiplied in the cystic epithelium, 3H-thymidine was given intraperitoneally to the mice 4 hr prior to the isolation of the cysts (3 and 6 days), which were then processed for autoradiography. As occurred in vivo (Fukuyama and Bernstein, 1961; Leblond, Greulich and Pereira, 19641, only cells in the basal layer of the cystic epithelium incorporated 3H-thymidine (Figure 2f). This result, plus the fact that mitotic figures are present in the basal layer, shows that cultured epidermal cells replicated at least to some extent in the subcutaneous environment. The histochemical properties of the cystic epithe-
Figure
1.
Cultured
Rabbit
Stratified
Squamous
Epithelial
Cells
6-9 day old primary cultures were fixed wtth formaldehyde and photographed under phase-contrast. (a) An epidermal colony. Cells surrounding the epidermal colony are lethally irradiated 3T3 fibroblasts. (b) A cornea1 epithelial colony. Note that the cells are more refractile and “fibroblastic” than epidermal and esophageal cells. (c) An esophageal epithelial colony. The epithelial nature of these three cell types has been demonstrated by their positive anti-keratin staming (Sun and Green, 1978a; Sun et al., 1980). Bar represents 50 pm.
lium and the in vivo epidermis were compared periodic acid-Schiff (PAS) staining, which detects cogen and other carbohydrates, and rhodamine which stains the stratum corneum (MacConaill Gurr, 1964). It was found that both cystic and in epithelia were negative with PAS and positive rhodamine B (Figures 2g and 2h).
by glyB, and vivo with
Regulation 19
Figure
of
Epithekal
2. Differentiation
Differentiation
of Cultured
Rabbit
Epidermal
Cells in Athymic
Mice
(a) Rabbit trunk skin, in viva. The epidermis is extremely thin, a common feature of furred animals. (b) A low magnification survey micrograph of a 5 day cyst formed by cultured rabbit epidermal cells. Note the viable epithelium lining the cystic cavity and the cell debris in the center. (ME) Mouse epidermis; (RE) rabbit (cystic) epidermis; (CD) cell debris. Bar represents 500 pm. (c)An epithelial cyst formed by cultured rabbit epidarmal cells two days after injection. Note the newly formed keratohyalin granules (arrows). (d) A cyst 3 days after injection. Note the well organized cell layers and the newly formed keratohyalin granules (G) and enucleated stratum corneum (SC; partial separation of the stratum corneum may have occurred during processing). (e) Cyst 34 days after injection. The main histological features [keratohyalin granules (G). stratum corneum (SC)] are similar to those of the day 3 sample. However, note the extremely thick stratum corneum layers and the relatively thin living cellular layers. (f) 3Hthymidine incorporation in the epidermal cyst. 4 hr before sacrifice, 125 MCi of ‘H-thymidine were injected intraperitoneally into an athymic mouse bearing a 3 day old epidermal nodule. A section of the cyst was treated with xylene, covered with Kodak NTB-2 emulsion and exposed at 4°C for 4 weeks before development. Note that in the cystic epithelium only basal cells are labeled (arrows). Labeled cells located beneath the cystic epithelium are presumably mouse mesenchymal in origin. Nuclear fast red-stained. (g. h) Rhodamine B staining of the in viva epidermis (g) and a 5 day epidermal cyst (h). Stratum corneum stains red (asterisks) whereas other Cells and tissues stain blue. All except (f-h) were stained with hematoxylin and eosin. Bars represent 25 pm for all figures except (b).
Cell 20
Morphological Differentiation of Cultured Epithelial Cells in Athymic Mice
Cornea1
When cultured rabbit cornea1 epithelial cells were injected subcutaneously into athymic mice, they formed a cyst lined with a stratified squamous, nonkeratinized, epithelium. Like the cornea1 epithelium in vivo, it lacked both keratohyalin granules and a stratum corneum (Figures 3a and 3b). Due to its high glycogen content (Smelser and Ozanics, 1953), normal cornea1 epithelium gives a diastase-sensitive reaction with PAS (Figures 3c and 3e). The epithelium of the cornea1 cysts behaved similarly (Figures 3d and 3f). Furthermore, consistent with the absence of a stratum corneum, both the cystic and in vivo cornea1 epithelia were negative when tested with rhodamine B.
Morphological Esophageal
Differentiation of Cultured Epithelial Cells in Athymic Mice
When injected into athymic mice, cultured rabbit esophageal epithelial cells formed a cyst lined with a parakeratinized epithelium (Figure 4b). It closely resembled the in vivo epithelium (Figure 4a) with respect to the organization of cellular layers, the absence of keratohyalin granules, the formation of an eosinophilic, nucleated stratum corneum, and positive PAS staining (diastase-resistant) of the superficial cells (Figures 4c and 4d).
Keratin Pattern as a Biochemical Epithelial Differentiation
Marker
of
Keratins are a group of water-insoluble proteins (40K68K) which form tonofilaments in epidermal cells and a wide variety of other epithelial cells (Sun and Green, 1977, 1978a; Franke et al., 1978, 1979; Sun, Shih and Green, 1979; Sun, Doran and Vidrich, 1980). These proteins are particularly abundant in stratified squamous epithelia. We analyzed the water-insoluble keratin fractions of intact rabbit skin, cornea1 and esophageal epithelia by SDS-gel electrophoresis (Figures 5a, 5d and 59). Rabbit epidermis showed two major (58K, 56K) and four minor (64K, 63K, 51K, 46K) keratin bands. Esophageal epithelium showed only two keratin bands (58K, 45K), in agreement with the observations of Milstone and McGuire (1978) while cornea1 epithelium gave two major (64K, 55K) and one minor (58K) keratin bands. Although the three epithelia may have some keratins in common, such as the 58K dalton component, their overall keratin patterns were distinct. The keratin pattern therefore seems useful as a biochemical marker of epithelial differentiation. When grown in culture, the three rabbit epithelia ceased to show distinctive keratin patterns and instead expressed a similar set of keratins (59K, 57K, 51 K, 46K, 44K; Figures 5b, 5e and 5h). An exception was that cornea1 cells possessed two extra water-
Figure 3. m Athymic
Differentiation Mice
of Cultured
Rabbit
Corneal
Epithelial
Cells
(a) Cornea. m VIVO. Hematoxylm and eosin (H 8 El-stained. (b) Cultured cornea1 epithelial cells in athymic mouse (a 7 day old cyst). Note the absence of keratohyalin granules and stratum corneum. as well as the presence of nuclei even in the most superficial cells. H 8 E-stained. (c) Cornea, m viva. PAS-stained. (d) Cystic epithelium formed by cultured cornea1 epithelial cells. PAS-stained. (e) Cornea. m VIVO. Dlastase-treated and then PAS-stained. (f) Cystic epithelium formed by cultured cornea1 epithelial cells. Diastaseand PASstamed. Bar represents 25 pm.
insoluble proteins (64K and 41 K; compare Sun and Green, 1977). Significantly, the keratin patterns of all three cultured cell types were different from their corresponding intact epithelia. Thus cultured epidermal cells lacked the two large keratins (64K, 63K) present in the epidermis in vivo. On the other hand, both cultured cornea1 and esophageal cells possessed more keratin bands than were originally present in the intact tissues (Figure 5). Subcutaneous cysts formed by cultured skin and esophageal epithelial cells reacquired their in vivo keratin patterns (Figures 5c and 5f). Cells of the epidermal cysts regained the high molecular weight keratins characteristic of the in vivo epidermis, and some proteins also reappeared in the 56K dalton region. The esophageal cysts contained two promi-
Regulation 21
of
Eplthellal
Differentlatlon
nent keratins (58K, 45K) similar to those found in the intact epithelium. Concomitant with the reappearance of this in vivo pattern was the marked diminution of those keratins previously found in cultured esophageal epithelial cells. Cornea1 epithelial cysts were exceptional in that their keratin pattern remained similar to that of the cultured cornea1 cells (Figure 5); perhaps related to this is the finding that the cornea1 cystic epithelium did not incorporate 3H-thymidine.
Discussion Mesenchymo-Epithelial Life
Interactions
in Post Natal
It has been well established that in embryonic tissues mesenchyme plays an important role not only in supporting the differentiation but also in actively controlling the developmental fate of its overlying epithelium (Auerbach, 1960; McLaughlin, 1963; Grobstein, 1967; Fleischmajer and Billingham, 1968; Kollar, 1972a, 1972b; Sengel, 1976; Saxen, 1977; Lash and Burger, 1977; Cunha and Lung, 1979). Experiments involving adult tissues have, however, generated a somewhat confusing picture as to whether specific mesenchymal instructions are continuously required for proper epithelial differentiation (Billingham and Silvers, 1963; Wessells, 1967). Using a transplantation approach, Billingham and Silvers (1967) found that when skin from guinea pig ear, sole and trunk was separated into epidermal and dermal components and heterotopic recombinants were studied, local dermis was found to be responsible for the characteristic mitotic rate, thickness and other minor histological variations of the overlying epidermis (Billingham and Silvers, 1967). The same investigators also observed that guinea pig tongue and esophageal epithelia were, perhaps somewhat unexpectedly, transformed into trunk epidermis when exposed to trunk dermis. Similar directive influences of the stroma were also reported after transplantation of human oral epithelia to dermis or oral mesenchyme (Van Scott and Reinertson, 1961; MacKenzie, Dabelsteen and Roed-Petersen, 1979) neonatal mouse uterine epithelium to cervical stroma (Cunha, 1976) mouse mammary epithelium to embryonic salivary mesenchyme (Sakakura, Sakagami and Nishizuka, 1979) and mouse bladder epithelium to embryonic urogenital sinus mesenchyme (Cunha, Lung and Reese, 1980). These results indicate that some adult epithelia may remain responsive to the directive influence from foreign mesenchyme, particularly stroma derived from related embryonic organs. In contrast, using a similar approach, it has also been shown that the histological specificities of several adult stratified squamous epithelia are faithfully conserved after they are placed on other anatomical sites. These experiments include the transplantation
I .‘s _lj Figure 4. Differenttahon Cells in Athymic Mice
of Cultured
Rabbit
Esophageal
Epithelial
(a) Esophageal epithelium, in VIVO. Hematoxylm and eosr (H 8 E)stamed (b) Cultured esophageal epithelial cells in an athymlc mouse (day 7). Note the formation of parakeratotic (nucleated) stratum corneum and the absence of keratohyalin granules. H 8 E-stained. (c) Esophageal epithelium. In VIVO. PAS-stained. (d) Cultured esophageal epithelial cyst. PAS-stained. Note the staining of the superflclal cells. Bar represents 25 pm.
of rabbit cornea1 epithelium to dermis (Billingham and Medawar, 1950), guinea pig tongue and esophageal epithelium to sole or ear dermis (Billingham and Silvers, 1967) rat epidermis to uterine stroma (Beer and Billingham, 1970) rat oral mucosa to ear dermis (Oliver, 1973) and mouse vaginal epithelium to uterine stroma (Cunha and Lung, 1979). Such results suggest that despite the morphological similarities of their basal cells, these different epithelia must have diverged from each other during development. Related to these phenomena, both the inductive power of the stroma and the responsiveness of the epithelia are known to decrease in an age-dependent fashion not
Cell 22
only during development 1976; Sengel, 1976).
but also after birth (Cunha,
number of cells increased 1 03-1 O4 fold during the in vitro growth, thus diluting out any original cellular components by at least the same factor. A related observation is that the life span of cellular “memory” following a previous exposure to an inductive event is usually short (Waddel and Soll, 1977; Levenson and Housman, 1979). It is therefore highly unlikely that any “controlling influence” derived originally from the subjacent mesenchyme remained active in our cultured epithelia.
Cell Culture-Athymic Mouse Approach As mentioned, transplantation experiments have yielded important information concerning mesenchymo-epithelial interactions. However, the interpretation of some of the data obtained by such experiments, particularly in cases where no marker was available for the unambiguous identification of the transplanted cells, may be seriously complicated by technical difficulties. These include the possible overgrowth of transplanted epithelium by remaining or adjacent host epithelial cells, the contamination of transplanted epithelium by original underlying mesenchymal tissues, the early rejection of the transplants due to infection or wound contraction, and the insufficient multiplication of the transplanted cells. To overcome these problems, we have adopted a new approach in which we grow various stratified squamous epithelial cells in culture and compare their differentiated properties under common in vitro and in vivo conditions. There are several advantages to this approach. First, using a feeder cell technique and EDTA treatment, there is practically no contamination of our epithelial preparations by the original fibroblasts or any other mesenchymal elements (Rheinwald and Green, 1975; Sun and Green, 1976). Second, the subcutaneous injection of disaggregated epithelial cells is relatively simple and reproducible, and alleviates the problems of wound contraction and overgrowth of transplanted cells by neighboring host epithelium. The reproducible morphological specificities of the cysts (in four independent experiments) following the injection of various epithelial cells, as well as the lack of cyst formation following the injection of medium alone, proved that the injected epithelial cells were responsible for the cyst formation. Third, the skin
Differentiated States of Cultured Epithelial Cells When grown in culture, rabbit skin, cornea1 and esophageal epithelial cells expressed numerous markers of differentiation and are therefore well differentiated as keratinocytes. However, all three cultured rabbit cell types lost some of their in vivo features and became less distinctive from each other both morphologically (Figure 1) and biochemically (Figure 5). Such an in vitro conversion to a similar phenotypic state is even more striking for cultured human skin and cornea1 epithelial cells, which are indistinguishable (Sun and Green, 1977) although both can also regain their morphological specificities upon injection into athymic mice (M. Jarvinen, I. M. Freedberg and T.-T. Sun, unpublished observation). The mechanism of such an in vitro conversion is not known. However, one may speculate that under the present culture conditions the various stratified squamous epithelia may have adopted more “primitive,” and thus less distinctive, programs of differentiation. Environmental Modulation Although cultured epidermal cells do not differentiate in a manner identical to that of the intact tissue (e. g. Figure 5) it was not clear whether this was due to an irreversible dedifferentiation of cultured epidermal cells (Moscona, 1964) to the in vitro selection of a
cornea
esoph.
Figure 5. Keratin Proteins of Rabbit Skin, Esophageal and Cornea1 Epithelial Cells
.,I
-66 -60
56“”
-40
a
bc
d
e
f
g
h
i
std.
The water-msoluble keratin fractions of various epithelial cells were resolved electrophoretically on a 15% SDS gel. Tracks (a, d and 9) are keratins of the epithelia in Go; (b, e and h) are cultured epithelial cells; (c, f and i) are 10 day old cysts formed by cultured epithelial cells in athymic mice.
;;gulation
of Epithelial
Differentiation
minor population of “abnormal” cells, or simply to the peculiarities of the in vitro growth environment. We have found that both primary cultures of rabbit and secondary cultures of human epidermal cells can reexpress their in vivo differentiated features when injected subcutaneously into athymic mice. The restored phenotypes of rabbit epidermal cells include the formation of granular cells and an enucleated stratum corneum (Figure 2d), the reacquisition of histochemical properties (Figures 2g and 2h), the high molecular weight keratins (Figure 5) and various ultrastructural features (R. Lavker and T.-T. Sun, manuscript in preparation). These results rule out the first two possibilities and strongly suggest that the differentiation of epidermal cells can be reversibly modulated by the external environment (compare Billingham and Silvers, 1967). Since a similar situation exists in cornea1 (Figure 3) and esophageal (Figures 4 and 5) epithelia, the external environment may under certain conditions play an important role in modulating the differentiation of stratified squamous epithelia. Although in subcutaneous sites cultured epidermal cells reacquired many of their in vivo features, these cells remained slightly different from the intact epidermis in two respects. Whereas under in vivo conditions it takes lo-14 days for a basal cell to become a stratum corneum cell, in epidermal cysts it took, at least initially, less than two days for granular cells (Figure 2c) and three days for the enucleated stratum corneum to form (Figure 2d). Although these cells could have been derived from newly replicated basal cells (Figure 2f), in view of the extreme rapidity of the cystic keratinization, it seems more probable that such differentiated cells originated from partially differentiated ones preexisting in culture. The other difference is that the viable cellular layers of the cystic epithelium, particularly in cysts less than 5-10 days old, were usually much thicker than the normal trunk epidermis (Figures 2a and 2d). In the older cysts, the thickness of the living epithelium was reduced (Figure 2e), presumably due to a rate of terminal differentiation exceeding that of cell replacement.
Intrinsic
Divergence
We have shown that rabbit skin, cornea1 and esophageal epithelia remained distinguishable even when provided with an identical in vitro (Figure 1) or in vivo (Figures 2, 3 and 4) environment. These results indicate that the three epithelia are not equipotential and must have diverged from each other during development. Since the distinctive in vivo phenotype of each epithelium was expressed when the cells were transplanted to the same subcutaneous site, the expression of these differences does not depend on specific mesenchymal instruction but on permissive factors not present in the culture system. Thus under in vivo
conditions intrinsic divergence must play a predominant role in determining the characteristic phenotypes of the epithelia. This conclusion is in general agreement with that of Billingham and Silvers (1967) and is particularly interesting in view of the fact that epidermal and cornea1 epithelial cells are both ectodermderived and closely related to each other embryologically (Coulombre, 1964; Coulombre and Coulombre, 1971).
Experimental
Procedures
Cell Culture Corneas from freshly killed rabbits (New Zealand White female, 4-6 weeks old) were minced finely, suspended in 0.2% trypsin. 0.003% EDTA (in saline). and stirred at 37°C for 30 min. The disaggregated cornea1 cells were grown in Dulbecco’s modified Eagle’s medium containmg 20% fetal calf serum and hydrocortisone (0.5 pg/ml) in the presence of lethally irradiated 3T3 fibroblasts as described (Rheinwald and Green, 1975: Sun and Green, 1977). To isolate epidermal cells a small piece of shaved rabbit skin (3 cm X 3 cm) was excised, mmced. trypsinized and grown as described above. Esophageal epithelial cells were isolated from a central portion of the esophagus (2 cm in length) which had been ligated at both ends and filled by syringe with the trypsin:EDTA mixture. After an incubation at 37°C for 30 min. epithelial cells were isolated by light scrapmg of the luminal surface and then grown as described above. Under these conditions, over 80-95% of the proliferating colonies in primary cultures of the three tissues were identified as eplthelial cells by indirect lmmunofluorescent staining with an ant+-keratin anhserum (Sun and Green, 1978a; Sun et al., 1980). The remaining colonies were keratin-negative and fibroblastic and could be selectlvely removed by an EDTA treatment (Rheinwald and Green, 1975: Sun and Green, 1976). Injection of Cultured Epithelial Cells into Athymic Mice After the 3T3 feeder cells and any contaminating rabbit fibroblasts were selectively removed by EDTA treatment, epithelial cells were trypsinized and resuspended in culture medium. Aliquots (0.1 ml) containing 5 X 1 O6 cells were injected subcutaneously into the flanks of athymic mice (EALB/c background, 4-6 weeks old, from SpragueDawley. Madison, Wisconsin). The soft swelling produced at the site of injection usually decreased slightly in size within 24-48 hr. presumably due to the absorptton of the medium. A small, firm nodule subsequently began to form. The size of the nodule either remained approximately the same or increased slightly during the ensumg two weeks to a maximum of 3-5 mm In diameter and then started to decrease slowly. Usually the nodules became unpalpable in 1-2 months. After various intervals, animals were sacrificed by cervical dislocation and the nodules were excised for histologtcal or biochemtcal analvses. Isolation of Tissues and Cells A broad area of rabbit skin (10 cm X 10 cm) was shaved, excised and epilated by hot wax treatment (Pusey, 1926). The skm surface was scraped with a scalpel blade to obtam the epidermal cells. Esophageal and cornea1 epithelial cells were similarly isolated by scraping the untreated surfaces of the tissues. To determine the completeness of the removal of epithelial cells, residual tissues were examined histologically. The removal ranged from almost complete for the cornea1 epithelium to partial for the esophageal epithelium includmg only about 10% of its basal layer. However, control experlments involving the separation of the epithelia from the underlying stroma by prior incubation of the tissues wtth 0.24 M NH&I or 0.25% trypsin followed by scrapmg yielded cells with essentially Identical keratin patterns.
Cell 24
Cultured epithelial cells were Isolated by scraping the dashes with a rubber polrceman. Cysts of the athymrc mace were excised surgically and freed of adhering tissues before processing. Keratin Analysis To remove the water-insoluble proteins, the cells or trssues were homogenized in buffer contammg 0.025 M Tris (pH 7.4). 0.001 M phenylmethylsulfonyl fluoride (PMSF) with a polytron (Tekmar Company, speed setting 50. 1 min) and then centrifuged at 12,000 X g at 4°C for 30 min. The extraction was repeated once and the waterinsoluble pellet was extracted with buffer containmg 0.025 M Tris (pH 7.4). 9 M urea, 0.05 M DTT and 0.001 M PMSF. The latter fraction, containing predominantly keratin proteins, was then analyzed on a Laemmli-type discontinuous 15% slab gel containing SDS (Sun and Green, 1978b). Acknowledgments The preliminary observation that cultured normal human epidermal cells keratinized when injected subcutaneously into athymic mouse was made by Howard Green and one of us (T.-T. S.) at MIT in the fall of 1977 and provided the stimulus for these investigations. We are grateful to Howard Green for critical reading of the manuscript, Lyle L. Sensenbrenner for the use of a cell Irradiator and Paula Bonitz. Lydia O’Neill and Koenraad Vandegaer for excellent technical assistance. These investigations were aided by grants from the NIH and the American Cancer Society. A.V. was a NIH postdoctoral tramee and T.-T. S. was the recipient of an NIH Research Career Development Award. The costs of publication of this article were defrayed m part by the payment of page charges. This article must therefore be hereby marked “advertisemenl” in accordance with 1.8 U.S.,C. Section 1734 solely to indicate this fact. Received
November
19. 1979; revrsed
July 24, 1980
Flerschmajer, R. and Billingham, R. E.. eds. (1968). Eprthelial-Mesenchymal Interactions. (Baltimore: Wrlliams and Wilkins Co ). Fogh, J. and Grovanella. Expenmental and Clinical Franke. Different rescence
B. C., eds. (1978). The Nude Research. (New York: Academic
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genes
m
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on epithelral
Milstone, L. M. and McGuire, J. S. (1978). Comparison of proteins that form 8-l 0 nm filaments in two keratinizing epithelia and endothelial cells in culture. J. Cell Brol. 79, CD1 48.
Billingham. R. E. and Silvers, W. K. (1963). The origin and conservation of epidermal specrfrcrtres. N. Eng. J. Med. 268, 539-545.
Moscona, A. A. (1964). Studies on stability of phenotypic traits in embryonic integumental tissues and cells. In The Epidermis, W. Montagna and W. C. Lobitz, Jr., eds. (New York: Academic Press), pp. 83-96.
Billingham, R. E. and Silvers, W. K. (1967). Studies on the conservation of epidermal specificities of skm and certain mucosa in adult mammals. J. Exp. Med. 125, 429-446.
Oliver, R. F. (1973). Response of oral eprthelium to the influence of whisker dermal papillae in the adult rat. Arch. Oral Biol. 78, 413421.
Coulombre, A. J. (1964). Problems in cornea1 morphogenesrs. Adv. Morphogen. 4, 81-l 09. Coulombre, J. L. and Coulombre. A. J. (1971). Metaplastic induction of scales and feathers m the cornea1 anterior epithelium of the chick embryo. Dev. Biol. 25, 464-478.
Pusey, W. A. (1926). 87, 663.
Cunha, G. R. (1976). Stromal induction and specification of morphogenesis and cytodifferentiation of the epithelia of the Mullerian ducts and urogenital sinus during development of the uterus and vagina in mice. J. Exp. Zool. 796, 361-370.
Sakakura, T.. Sakagami, Y. and Nishizuka. Y. (1979). Persistence of responsiveness of adult mouse mammary gland to induction by embryonic mesenchyme. Dev. Biol. 72, 201-210.
Cunha, G. R. and Lung, B. (1979). The importance morphogenesis and functional activity of urogenital Vitro 15. 50-71.
of stroma epithelium.
in In
Cunha, G. R., Lung, B. and Reese, B. (1980). Glandular epithelial induction by embryonic mesenchyme in adult bladder epithelium Of BALB/c mice. Invest. Ural. 17, 302-304.
Recipe
for eprlating
wax.
J. Am. Med. Assoc.
Rheinwald, J. R. and Green, H. (1975). Serial cultrvation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell 6, 331-343.
Saxen, L. (1977). Directive versus permissive induction: hypothesis. In Cell and Tissue Interactions, J. W. Lash Burger, eds. (New York: Raven Press), pp. l-9.
a working and M. M.
Sengel, P. (1976). University Press).
Cambridge
Morphogenesis
of Skin.
(London:
Smelser. G. K. and Ozanics, V. (1953). Structural changes m corneas of guinea pigs after wearing contact lenses. Arch. Ophthalmol. 49,
Regulation 25
of
Eprthelial
Differentration
335-340 S&inert. P. and Yuspa. keratinization by mouse 1491-1493. Sun, T.-T. keratinocyte
S. H. (1978). Biochemrcal epidermal cells rn culture.
ewdence for Science 200.
and Green, H. (1976). Differentiation of the epidermal in cell culture: formation of the cormfied envelope. Cell
9. 51 l-521. Sun, T.-T. and Green, H. (1977). Cultured eptthelial cells of cornea, conjunctiva and skin: absence of marked intrinsic divergence of thew differentiated states. Nature 269, 489-493. Sun,
T.-T.
keratin
and
fibers
Green,
in cultured
H. (1978a). ceils.
lmmunofluorescent
staining
Sun. T.-T. and Green, H. (1978b). Keratin filaments of cultured epidermal cells: formation of intermolecular disulfide bonds terminal
differentiation.
of
Cell 14, 469-476.
J. Biol. Chem.
253,
human durmg
2053-2060.
Sun, T.-T., Shih, C. and Green, H. (1979). Keratin cytoskeleton epithelial cells of internal organs. Proc. Nat. Acad. Sci. USA 2813-2817.
rn 76,
Sun, T.-T., Doran. T. I. and Vidrich. A. (1980). The use of anti-kerattn antibodies for the identification of cultured epithelial cells. In In Vitro Epithelia and Birth Defects, XV/(Z), B. S. Danes. ed. (New York: Alan R. Liss for the March 196.
of Dimes
Birth
Defects
Foundation).
pp. 183-
Van Scott, E. J. and Reinertson. R. P. (1961). The modulating influence of stromal environment on epithelial cells studied in human autotransplants. J. Invest. Dermatol. 36, 109-l 31. Waddel. D. R. and Soll. D. R. (1977). Acharacterization phenomenon in Dictyostelium. Dev. Biol. 60, 83-92. Wessells, derivatives.
N. K. (1967). Differe-tion N. Eng. J. Med. 277, 2+-33.
of epidermis
of the erasure
and
epidermal
17-25.
November
1980
(Part
1). Copyright
0 1960
by MIT
Intrinsic and Extrinsic Regulation of the Differentiation of Skin, Cornea1 and Esophageal Epithelial Cells Thomas
I. Doran,
Alda Vidrich
Departments of Dermatology, and Anatomy The Johns Hopkins University Baltimore, Maryland 21205
and Tung-Tien and
of Cell
School
Sun*
Biology
of Medicine
Summary Basal cells of the stratified squamous epithelia of rabbit skin, cornea and esophagus appear morphologically similar. However, the histological features of their subsequent differentiation are different, and the three epithelia are characterized by distinctive keratin proteins. To analyze the relative importance of intrinsic versus extrinsic factors in regulating the differentiation of these epithelia, we compared their behavior under identical in vitro and in vivo conditions. When cultured in the presence of 3T3 feeder cells, keratinocytes from all three epithelia formed differentiating colonies. Although in culture the three cell types approached a common phenotype, they remained distinguishable morphologically and, in some cases, biochemically. When these cultured epithelial cells were trypsinized, suspended in medium and injected subcutaneously into athymic (nude) mice, each of the three cell types generated a characteristic cyst consisting of stratified squamous epithelium. Cultured skin, cornea1 and esophageal keratinocytes formed epithelia which were keratinized, nonkeratinized and parakeratinized, respectively. In addition, the injected skin and esophageal epithelial cells reacquired their distinctive in vivo keratin patterns. These data suggest that the three epithelia are not equipotential. Furthermore, since the distinctive in vivo phenotype of each epithelium was expressed when the cells were transplanted to the same subcutaneous site, the expression of these differences does not depend on specific mesenchymal instruction but on permissive factors not present in the culture system. Thus under in vivo conditions intrinsic divergence must play a predominant role in determining the characteristic phenotypes of the three epithelia. On the other hand, the finding that the morphological and biochemical differentiation of a given epithelium can be reversibly modulated by the external environment demonstrates that extrinsic factors may, under certain conditions, also play a role in regulating epithelial differentiation.
These multiple layered epithelia are composed mainly of keratinocytes which contain abundant keratin filaments (Sun and Green, 1977, 1978a). Although the basal cells of different stratified squamous epithelia appear morphologically similar, their subsequent differentiation can be very different; hence the descriptions of keratinized epidermis, nonkeratinized cornea1 epithelium and parakeratinized (rabbit) esophageal epithelium (Alvares and Meyer, 1971). Normally the histological features of these various epithelia are faithfully conserved throughout post natal life. To explain such a conservation of tissue specificity, two hypotheses, which are not mutally exclusive, have been proposed. First, despite their apparent similarities, the basal cells of different epithelia may have diverged irreversibly from each other during development (intrinsic divergence), thus resulting in different programs of differentiation. Alternatively, basal cells of different stratified squamous epithelia may be equipotential and therefore identical to each other (Billingham and Silvers, 1963). Their distinctive in vivo differentiation would then be due to different local, directive influences derived from their environment, for example, from the underlying mesenchymal tissues (external modulation; for reviews of studies using a transplantation approach, see Billingham and Silvers, 1963, 1967; Wessells, 1967). In the presence of lethally irradiated 3T3 feeder cells, epidermal cells undergo clonal growth and retain several important in vivo differentiation markers (Rheinwald and Green, 1975; Sun and Green, 1976, 1978a, 1978b). However, their differentiation is not identical to that of the intact epidermis, since neither keratohyalin granules nor an enucleated stratum corneum are formed. Furthermore, cultured epidermal cells lack the largest keratin proteins (63K-68K daltons) found in the epidermis in vivo (Fuchs and Green, 1978, 1980; Steinert and Yuspa, 1978; Sun and Green, 1978b). In this paper we show that cultured epidermal cells regained their in vivo phenotypes when placed subcutaneously in athymic (nude) mice. The same subcutaneous environment also allowed cultured cornea1 and esophageal epithelial cells to express their distinctive in vivo patterns of differentiation. These results suggest that the three epithelia in (juvenile) animals are intrinsically different from each other and represent different cell lineages.
Results
Introduction
In Vitro
Differentiation
Stratified cornea,
Cultured lial cells fibroblast squamous proteins
rabbit skin, cornea1 and esophageal epithebehaved similarly: all three required suitable support for clonal growth, formed stratified structures and synthesized similar keratin (see below). However, consistent with pre-
squamous esophagus
epithelia line and several
the surfaces of skin, other body sites.
l TO whom reprint requests should be addressed Johns Hopkins Medical School, Baltimore, Maryland
at: Blalock 21205.
926,
of Rabbit
Epithelial
Cells
Cell 18
vious findings (Sun and Green, 1977). the three cultured rabbit cell types remained distinguishable from each other by several criteria. Minor but reproducible morphological differences could be discerned, particularly in colonies of fewer than -lo3 cells (Figures 1 a-l c). Cornified envelopes, a terminal differentiation product of some keratinocytes (Sun and Green, 1976), were formed by surface-cultured rabbit skin and esophageal epithelial cells, but not by cornea1 epithelial cells (compare Sun and Green, 1977). In addition, cornea1 cells elaborated two proteins (64K and 41K) not present in either epidermal (Sun and Green, 1977) or esophageal cells (see below). To examine whether such cultured epithelial cells retained their ability to differentiate normally in an in vivo environment, we studied their behavior in athymic mice, which are immunodeficient and thus can tolerate heterotransplants (Fogh and Giovanella, 1978). Morphological Differentiation of Cultured Epidermal Cells in Athymic Mice Primary cultures (1 O-l 6 days old) of rabbit epidermal cells were trypsinized and aliquots containing 5 x 1 O6 suspended cells were injected subcutaneously into the flanks of athymic mice (Figure 2b). Within two days these cells formed cysts (2-4 mm in diameter) with an epithelial structure lining the cavity and some cell debris in the center. Keratohyalin granules, previously absent in culture, appeared in the cystic epithelium (Figure 2~). The epithelium in day 3 cysts (Figure 2d) became similar to the keratinized epidermis in vivo; it had a well organized stratified squamous structure, prominent keratohyalin granules and an enucleated stratum corneum (Figure 2d). After day 3 the histological features of the cystic epithelium remained essentially unchanged, except for an increase in the number of stratum corneum layers and a decrease in that of living layers (Figure 3e). A similar formation of keratinized cysts (in shortterm experiments of up to 5 days) was observed when cultured epidermal cells were injected subcutaneously into normal BALB/c instead of athymic mice. Thus the results described were not due to conditions peculiar to the athymic nature of the recipient mice. To determine whether cells multiplied in the cystic epithelium, 3H-thymidine was given intraperitoneally to the mice 4 hr prior to the isolation of the cysts (3 and 6 days), which were then processed for autoradiography. As occurred in vivo (Fukuyama and Bernstein, 1961; Leblond, Greulich and Pereira, 19641, only cells in the basal layer of the cystic epithelium incorporated 3H-thymidine (Figure 2f). This result, plus the fact that mitotic figures are present in the basal layer, shows that cultured epidermal cells replicated at least to some extent in the subcutaneous environment. The histochemical properties of the cystic epithe-
Figure
1.
Cultured
Rabbit
Stratified
Squamous
Epithelial
Cells
6-9 day old primary cultures were fixed wtth formaldehyde and photographed under phase-contrast. (a) An epidermal colony. Cells surrounding the epidermal colony are lethally irradiated 3T3 fibroblasts. (b) A cornea1 epithelial colony. Note that the cells are more refractile and “fibroblastic” than epidermal and esophageal cells. (c) An esophageal epithelial colony. The epithelial nature of these three cell types has been demonstrated by their positive anti-keratin staming (Sun and Green, 1978a; Sun et al., 1980). Bar represents 50 pm.
lium and the in vivo epidermis were compared periodic acid-Schiff (PAS) staining, which detects cogen and other carbohydrates, and rhodamine which stains the stratum corneum (MacConaill Gurr, 1964). It was found that both cystic and in epithelia were negative with PAS and positive rhodamine B (Figures 2g and 2h).
by glyB, and vivo with
Regulation 19
Figure
of
Epithekal
2. Differentiation
Differentiation
of Cultured
Rabbit
Epidermal
Cells in Athymic
Mice
(a) Rabbit trunk skin, in viva. The epidermis is extremely thin, a common feature of furred animals. (b) A low magnification survey micrograph of a 5 day cyst formed by cultured rabbit epidermal cells. Note the viable epithelium lining the cystic cavity and the cell debris in the center. (ME) Mouse epidermis; (RE) rabbit (cystic) epidermis; (CD) cell debris. Bar represents 500 pm. (c)An epithelial cyst formed by cultured rabbit epidarmal cells two days after injection. Note the newly formed keratohyalin granules (arrows). (d) A cyst 3 days after injection. Note the well organized cell layers and the newly formed keratohyalin granules (G) and enucleated stratum corneum (SC; partial separation of the stratum corneum may have occurred during processing). (e) Cyst 34 days after injection. The main histological features [keratohyalin granules (G). stratum corneum (SC)] are similar to those of the day 3 sample. However, note the extremely thick stratum corneum layers and the relatively thin living cellular layers. (f) 3Hthymidine incorporation in the epidermal cyst. 4 hr before sacrifice, 125 MCi of ‘H-thymidine were injected intraperitoneally into an athymic mouse bearing a 3 day old epidermal nodule. A section of the cyst was treated with xylene, covered with Kodak NTB-2 emulsion and exposed at 4°C for 4 weeks before development. Note that in the cystic epithelium only basal cells are labeled (arrows). Labeled cells located beneath the cystic epithelium are presumably mouse mesenchymal in origin. Nuclear fast red-stained. (g. h) Rhodamine B staining of the in viva epidermis (g) and a 5 day epidermal cyst (h). Stratum corneum stains red (asterisks) whereas other Cells and tissues stain blue. All except (f-h) were stained with hematoxylin and eosin. Bars represent 25 pm for all figures except (b).
Cell 20
Morphological Differentiation of Cultured Epithelial Cells in Athymic Mice
Cornea1
When cultured rabbit cornea1 epithelial cells were injected subcutaneously into athymic mice, they formed a cyst lined with a stratified squamous, nonkeratinized, epithelium. Like the cornea1 epithelium in vivo, it lacked both keratohyalin granules and a stratum corneum (Figures 3a and 3b). Due to its high glycogen content (Smelser and Ozanics, 1953), normal cornea1 epithelium gives a diastase-sensitive reaction with PAS (Figures 3c and 3e). The epithelium of the cornea1 cysts behaved similarly (Figures 3d and 3f). Furthermore, consistent with the absence of a stratum corneum, both the cystic and in vivo cornea1 epithelia were negative when tested with rhodamine B.
Morphological Esophageal
Differentiation of Cultured Epithelial Cells in Athymic Mice
When injected into athymic mice, cultured rabbit esophageal epithelial cells formed a cyst lined with a parakeratinized epithelium (Figure 4b). It closely resembled the in vivo epithelium (Figure 4a) with respect to the organization of cellular layers, the absence of keratohyalin granules, the formation of an eosinophilic, nucleated stratum corneum, and positive PAS staining (diastase-resistant) of the superficial cells (Figures 4c and 4d).
Keratin Pattern as a Biochemical Epithelial Differentiation
Marker
of
Keratins are a group of water-insoluble proteins (40K68K) which form tonofilaments in epidermal cells and a wide variety of other epithelial cells (Sun and Green, 1977, 1978a; Franke et al., 1978, 1979; Sun, Shih and Green, 1979; Sun, Doran and Vidrich, 1980). These proteins are particularly abundant in stratified squamous epithelia. We analyzed the water-insoluble keratin fractions of intact rabbit skin, cornea1 and esophageal epithelia by SDS-gel electrophoresis (Figures 5a, 5d and 59). Rabbit epidermis showed two major (58K, 56K) and four minor (64K, 63K, 51K, 46K) keratin bands. Esophageal epithelium showed only two keratin bands (58K, 45K), in agreement with the observations of Milstone and McGuire (1978) while cornea1 epithelium gave two major (64K, 55K) and one minor (58K) keratin bands. Although the three epithelia may have some keratins in common, such as the 58K dalton component, their overall keratin patterns were distinct. The keratin pattern therefore seems useful as a biochemical marker of epithelial differentiation. When grown in culture, the three rabbit epithelia ceased to show distinctive keratin patterns and instead expressed a similar set of keratins (59K, 57K, 51 K, 46K, 44K; Figures 5b, 5e and 5h). An exception was that cornea1 cells possessed two extra water-
Figure 3. m Athymic
Differentiation Mice
of Cultured
Rabbit
Corneal
Epithelial
Cells
(a) Cornea. m VIVO. Hematoxylm and eosin (H 8 El-stained. (b) Cultured cornea1 epithelial cells in athymic mouse (a 7 day old cyst). Note the absence of keratohyalin granules and stratum corneum. as well as the presence of nuclei even in the most superficial cells. H 8 E-stained. (c) Cornea, m viva. PAS-stained. (d) Cystic epithelium formed by cultured cornea1 epithelial cells. PAS-stained. (e) Cornea. m VIVO. Dlastase-treated and then PAS-stained. (f) Cystic epithelium formed by cultured cornea1 epithelial cells. Diastaseand PASstamed. Bar represents 25 pm.
insoluble proteins (64K and 41 K; compare Sun and Green, 1977). Significantly, the keratin patterns of all three cultured cell types were different from their corresponding intact epithelia. Thus cultured epidermal cells lacked the two large keratins (64K, 63K) present in the epidermis in vivo. On the other hand, both cultured cornea1 and esophageal cells possessed more keratin bands than were originally present in the intact tissues (Figure 5). Subcutaneous cysts formed by cultured skin and esophageal epithelial cells reacquired their in vivo keratin patterns (Figures 5c and 5f). Cells of the epidermal cysts regained the high molecular weight keratins characteristic of the in vivo epidermis, and some proteins also reappeared in the 56K dalton region. The esophageal cysts contained two promi-
Regulation 21
of
Eplthellal
Differentlatlon
nent keratins (58K, 45K) similar to those found in the intact epithelium. Concomitant with the reappearance of this in vivo pattern was the marked diminution of those keratins previously found in cultured esophageal epithelial cells. Cornea1 epithelial cysts were exceptional in that their keratin pattern remained similar to that of the cultured cornea1 cells (Figure 5); perhaps related to this is the finding that the cornea1 cystic epithelium did not incorporate 3H-thymidine.
Discussion Mesenchymo-Epithelial Life
Interactions
in Post Natal
It has been well established that in embryonic tissues mesenchyme plays an important role not only in supporting the differentiation but also in actively controlling the developmental fate of its overlying epithelium (Auerbach, 1960; McLaughlin, 1963; Grobstein, 1967; Fleischmajer and Billingham, 1968; Kollar, 1972a, 1972b; Sengel, 1976; Saxen, 1977; Lash and Burger, 1977; Cunha and Lung, 1979). Experiments involving adult tissues have, however, generated a somewhat confusing picture as to whether specific mesenchymal instructions are continuously required for proper epithelial differentiation (Billingham and Silvers, 1963; Wessells, 1967). Using a transplantation approach, Billingham and Silvers (1967) found that when skin from guinea pig ear, sole and trunk was separated into epidermal and dermal components and heterotopic recombinants were studied, local dermis was found to be responsible for the characteristic mitotic rate, thickness and other minor histological variations of the overlying epidermis (Billingham and Silvers, 1967). The same investigators also observed that guinea pig tongue and esophageal epithelia were, perhaps somewhat unexpectedly, transformed into trunk epidermis when exposed to trunk dermis. Similar directive influences of the stroma were also reported after transplantation of human oral epithelia to dermis or oral mesenchyme (Van Scott and Reinertson, 1961; MacKenzie, Dabelsteen and Roed-Petersen, 1979) neonatal mouse uterine epithelium to cervical stroma (Cunha, 1976) mouse mammary epithelium to embryonic salivary mesenchyme (Sakakura, Sakagami and Nishizuka, 1979) and mouse bladder epithelium to embryonic urogenital sinus mesenchyme (Cunha, Lung and Reese, 1980). These results indicate that some adult epithelia may remain responsive to the directive influence from foreign mesenchyme, particularly stroma derived from related embryonic organs. In contrast, using a similar approach, it has also been shown that the histological specificities of several adult stratified squamous epithelia are faithfully conserved after they are placed on other anatomical sites. These experiments include the transplantation
I .‘s _lj Figure 4. Differenttahon Cells in Athymic Mice
of Cultured
Rabbit
Esophageal
Epithelial
(a) Esophageal epithelium, in VIVO. Hematoxylm and eosr (H 8 E)stamed (b) Cultured esophageal epithelial cells in an athymlc mouse (day 7). Note the formation of parakeratotic (nucleated) stratum corneum and the absence of keratohyalin granules. H 8 E-stained. (c) Esophageal epithelium. In VIVO. PAS-stained. (d) Cultured esophageal epithelial cyst. PAS-stained. Note the staining of the superflclal cells. Bar represents 25 pm.
of rabbit cornea1 epithelium to dermis (Billingham and Medawar, 1950), guinea pig tongue and esophageal epithelium to sole or ear dermis (Billingham and Silvers, 1967) rat epidermis to uterine stroma (Beer and Billingham, 1970) rat oral mucosa to ear dermis (Oliver, 1973) and mouse vaginal epithelium to uterine stroma (Cunha and Lung, 1979). Such results suggest that despite the morphological similarities of their basal cells, these different epithelia must have diverged from each other during development. Related to these phenomena, both the inductive power of the stroma and the responsiveness of the epithelia are known to decrease in an age-dependent fashion not
Cell 22
only during development 1976; Sengel, 1976).
but also after birth (Cunha,
number of cells increased 1 03-1 O4 fold during the in vitro growth, thus diluting out any original cellular components by at least the same factor. A related observation is that the life span of cellular “memory” following a previous exposure to an inductive event is usually short (Waddel and Soll, 1977; Levenson and Housman, 1979). It is therefore highly unlikely that any “controlling influence” derived originally from the subjacent mesenchyme remained active in our cultured epithelia.
Cell Culture-Athymic Mouse Approach As mentioned, transplantation experiments have yielded important information concerning mesenchymo-epithelial interactions. However, the interpretation of some of the data obtained by such experiments, particularly in cases where no marker was available for the unambiguous identification of the transplanted cells, may be seriously complicated by technical difficulties. These include the possible overgrowth of transplanted epithelium by remaining or adjacent host epithelial cells, the contamination of transplanted epithelium by original underlying mesenchymal tissues, the early rejection of the transplants due to infection or wound contraction, and the insufficient multiplication of the transplanted cells. To overcome these problems, we have adopted a new approach in which we grow various stratified squamous epithelial cells in culture and compare their differentiated properties under common in vitro and in vivo conditions. There are several advantages to this approach. First, using a feeder cell technique and EDTA treatment, there is practically no contamination of our epithelial preparations by the original fibroblasts or any other mesenchymal elements (Rheinwald and Green, 1975; Sun and Green, 1976). Second, the subcutaneous injection of disaggregated epithelial cells is relatively simple and reproducible, and alleviates the problems of wound contraction and overgrowth of transplanted cells by neighboring host epithelium. The reproducible morphological specificities of the cysts (in four independent experiments) following the injection of various epithelial cells, as well as the lack of cyst formation following the injection of medium alone, proved that the injected epithelial cells were responsible for the cyst formation. Third, the skin
Differentiated States of Cultured Epithelial Cells When grown in culture, rabbit skin, cornea1 and esophageal epithelial cells expressed numerous markers of differentiation and are therefore well differentiated as keratinocytes. However, all three cultured rabbit cell types lost some of their in vivo features and became less distinctive from each other both morphologically (Figure 1) and biochemically (Figure 5). Such an in vitro conversion to a similar phenotypic state is even more striking for cultured human skin and cornea1 epithelial cells, which are indistinguishable (Sun and Green, 1977) although both can also regain their morphological specificities upon injection into athymic mice (M. Jarvinen, I. M. Freedberg and T.-T. Sun, unpublished observation). The mechanism of such an in vitro conversion is not known. However, one may speculate that under the present culture conditions the various stratified squamous epithelia may have adopted more “primitive,” and thus less distinctive, programs of differentiation. Environmental Modulation Although cultured epidermal cells do not differentiate in a manner identical to that of the intact tissue (e. g. Figure 5) it was not clear whether this was due to an irreversible dedifferentiation of cultured epidermal cells (Moscona, 1964) to the in vitro selection of a
cornea
esoph.
Figure 5. Keratin Proteins of Rabbit Skin, Esophageal and Cornea1 Epithelial Cells
.,I
-66 -60
56“”
-40
a
bc
d
e
f
g
h
i
std.
The water-msoluble keratin fractions of various epithelial cells were resolved electrophoretically on a 15% SDS gel. Tracks (a, d and 9) are keratins of the epithelia in Go; (b, e and h) are cultured epithelial cells; (c, f and i) are 10 day old cysts formed by cultured epithelial cells in athymic mice.
;;gulation
of Epithelial
Differentiation
minor population of “abnormal” cells, or simply to the peculiarities of the in vitro growth environment. We have found that both primary cultures of rabbit and secondary cultures of human epidermal cells can reexpress their in vivo differentiated features when injected subcutaneously into athymic mice. The restored phenotypes of rabbit epidermal cells include the formation of granular cells and an enucleated stratum corneum (Figure 2d), the reacquisition of histochemical properties (Figures 2g and 2h), the high molecular weight keratins (Figure 5) and various ultrastructural features (R. Lavker and T.-T. Sun, manuscript in preparation). These results rule out the first two possibilities and strongly suggest that the differentiation of epidermal cells can be reversibly modulated by the external environment (compare Billingham and Silvers, 1967). Since a similar situation exists in cornea1 (Figure 3) and esophageal (Figures 4 and 5) epithelia, the external environment may under certain conditions play an important role in modulating the differentiation of stratified squamous epithelia. Although in subcutaneous sites cultured epidermal cells reacquired many of their in vivo features, these cells remained slightly different from the intact epidermis in two respects. Whereas under in vivo conditions it takes lo-14 days for a basal cell to become a stratum corneum cell, in epidermal cysts it took, at least initially, less than two days for granular cells (Figure 2c) and three days for the enucleated stratum corneum to form (Figure 2d). Although these cells could have been derived from newly replicated basal cells (Figure 2f), in view of the extreme rapidity of the cystic keratinization, it seems more probable that such differentiated cells originated from partially differentiated ones preexisting in culture. The other difference is that the viable cellular layers of the cystic epithelium, particularly in cysts less than 5-10 days old, were usually much thicker than the normal trunk epidermis (Figures 2a and 2d). In the older cysts, the thickness of the living epithelium was reduced (Figure 2e), presumably due to a rate of terminal differentiation exceeding that of cell replacement.
Intrinsic
Divergence
We have shown that rabbit skin, cornea1 and esophageal epithelia remained distinguishable even when provided with an identical in vitro (Figure 1) or in vivo (Figures 2, 3 and 4) environment. These results indicate that the three epithelia are not equipotential and must have diverged from each other during development. Since the distinctive in vivo phenotype of each epithelium was expressed when the cells were transplanted to the same subcutaneous site, the expression of these differences does not depend on specific mesenchymal instruction but on permissive factors not present in the culture system. Thus under in vivo
conditions intrinsic divergence must play a predominant role in determining the characteristic phenotypes of the epithelia. This conclusion is in general agreement with that of Billingham and Silvers (1967) and is particularly interesting in view of the fact that epidermal and cornea1 epithelial cells are both ectodermderived and closely related to each other embryologically (Coulombre, 1964; Coulombre and Coulombre, 1971).
Experimental
Procedures
Cell Culture Corneas from freshly killed rabbits (New Zealand White female, 4-6 weeks old) were minced finely, suspended in 0.2% trypsin. 0.003% EDTA (in saline). and stirred at 37°C for 30 min. The disaggregated cornea1 cells were grown in Dulbecco’s modified Eagle’s medium containmg 20% fetal calf serum and hydrocortisone (0.5 pg/ml) in the presence of lethally irradiated 3T3 fibroblasts as described (Rheinwald and Green, 1975: Sun and Green, 1977). To isolate epidermal cells a small piece of shaved rabbit skin (3 cm X 3 cm) was excised, mmced. trypsinized and grown as described above. Esophageal epithelial cells were isolated from a central portion of the esophagus (2 cm in length) which had been ligated at both ends and filled by syringe with the trypsin:EDTA mixture. After an incubation at 37°C for 30 min. epithelial cells were isolated by light scrapmg of the luminal surface and then grown as described above. Under these conditions, over 80-95% of the proliferating colonies in primary cultures of the three tissues were identified as eplthelial cells by indirect lmmunofluorescent staining with an ant+-keratin anhserum (Sun and Green, 1978a; Sun et al., 1980). The remaining colonies were keratin-negative and fibroblastic and could be selectlvely removed by an EDTA treatment (Rheinwald and Green, 1975: Sun and Green, 1976). Injection of Cultured Epithelial Cells into Athymic Mice After the 3T3 feeder cells and any contaminating rabbit fibroblasts were selectively removed by EDTA treatment, epithelial cells were trypsinized and resuspended in culture medium. Aliquots (0.1 ml) containing 5 X 1 O6 cells were injected subcutaneously into the flanks of athymic mice (EALB/c background, 4-6 weeks old, from SpragueDawley. Madison, Wisconsin). The soft swelling produced at the site of injection usually decreased slightly in size within 24-48 hr. presumably due to the absorptton of the medium. A small, firm nodule subsequently began to form. The size of the nodule either remained approximately the same or increased slightly during the ensumg two weeks to a maximum of 3-5 mm In diameter and then started to decrease slowly. Usually the nodules became unpalpable in 1-2 months. After various intervals, animals were sacrificed by cervical dislocation and the nodules were excised for histologtcal or biochemtcal analvses. Isolation of Tissues and Cells A broad area of rabbit skin (10 cm X 10 cm) was shaved, excised and epilated by hot wax treatment (Pusey, 1926). The skm surface was scraped with a scalpel blade to obtam the epidermal cells. Esophageal and cornea1 epithelial cells were similarly isolated by scraping the untreated surfaces of the tissues. To determine the completeness of the removal of epithelial cells, residual tissues were examined histologically. The removal ranged from almost complete for the cornea1 epithelium to partial for the esophageal epithelium includmg only about 10% of its basal layer. However, control experlments involving the separation of the epithelia from the underlying stroma by prior incubation of the tissues wtth 0.24 M NH&I or 0.25% trypsin followed by scrapmg yielded cells with essentially Identical keratin patterns.
Cell 24
Cultured epithelial cells were Isolated by scraping the dashes with a rubber polrceman. Cysts of the athymrc mace were excised surgically and freed of adhering tissues before processing. Keratin Analysis To remove the water-insoluble proteins, the cells or trssues were homogenized in buffer contammg 0.025 M Tris (pH 7.4). 0.001 M phenylmethylsulfonyl fluoride (PMSF) with a polytron (Tekmar Company, speed setting 50. 1 min) and then centrifuged at 12,000 X g at 4°C for 30 min. The extraction was repeated once and the waterinsoluble pellet was extracted with buffer containmg 0.025 M Tris (pH 7.4). 9 M urea, 0.05 M DTT and 0.001 M PMSF. The latter fraction, containing predominantly keratin proteins, was then analyzed on a Laemmli-type discontinuous 15% slab gel containing SDS (Sun and Green, 1978b). Acknowledgments The preliminary observation that cultured normal human epidermal cells keratinized when injected subcutaneously into athymic mouse was made by Howard Green and one of us (T.-T. S.) at MIT in the fall of 1977 and provided the stimulus for these investigations. We are grateful to Howard Green for critical reading of the manuscript, Lyle L. Sensenbrenner for the use of a cell Irradiator and Paula Bonitz. Lydia O’Neill and Koenraad Vandegaer for excellent technical assistance. These investigations were aided by grants from the NIH and the American Cancer Society. A.V. was a NIH postdoctoral tramee and T.-T. S. was the recipient of an NIH Research Career Development Award. The costs of publication of this article were defrayed m part by the payment of page charges. This article must therefore be hereby marked “advertisemenl” in accordance with 1.8 U.S.,C. Section 1734 solely to indicate this fact. Received
November
19. 1979; revrsed
July 24, 1980
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