Cell Proliferation and Cell Differentiation in Tissue Cultures of Adult Muco-ciliary Epithelia*

Cell Proliferation and Cell Differentiation in Tissue Cultures of Adult Muco-ciliary Epithelia* A. C. NEVO, Z. WEISMAN and J. SADE Polymer Department The Weizmann Institute of Science, Rehovot, and the Ear, Nose and Throat Department, Meir Hospital, Kfar Saba, Israel

Received April-l974/Revised October 1974

Explants and monolayers from a variety of muco-ciliary epithelia were cultivated in vitro and the kinetics of their proliferation and differentiation described. New epithelial lining and epithelial-like monolayer sheets of cells formed, in which the migration cells were all originally undifferentiated cycling stem cells. They divided and differentiated in ML growth into cell types characteristic of the tissue source; however, the control mechanisms which regulate cell division and cell differentiation in the tissues were lost outside the tissue framework. Cell division and cyto-differentiation in ML growths both in ciliated and in mucus-producing cells, were not always mutually exclusive.

Introduction

The muco-ciliary (MC) epithelia ofthe respiratory system, including the middle ear, consist essentially of an outer layer of ciliated cells and of mucus producing ,,goblet" cells abovealayer ofstem cells. Theepitheliumis supportedby a layer ofcollagenous connective tissue and separated fromit by a basement membrane (Fig. 1). Above the epithelium thereisathinlayerofmucus, secretedbythegobeltcellsand (often) by subepithelial mucus glands. For the proper functioning of the system,-i.e. transport and clearance of the mucus by the cilia,-the rate of mucus secretionmustberegulated, as has beenshown by us in a previous study I 11. As the adult epithelial tissues are renewal systems in steady state condition, there must also exist control mechanisms which regulate both the rate of stem cell division and the kind of differentiation they will undergo. Excess of mucus production and failure of its clearance in cases of chronic and secretory otitis media [2, 3,41 and in chronic bronchitis [51 reflect a failure of the control of cell differentiation and of mucus secretion,

whatever the original pathogenic agent may be. These pathologic conditions are characterised by hypersecretion of mucus associated with a great increase of mucus-producing cells and a corresponding decrease of ciliated cells, at times associated paradoxically with metaplastic development of keratinising stratified squamous epithelium as shown in the middle ear by Sad&161. The purpose ofthe present work was to obtain suitable in vitro model systems for investigation of factors and mechanisms which induce and control cell proliferation and cell differentiation in respiratory epithelia, with implications to the pathological conditions and metaplasia described above. The experimental systems used were tissue explants and monolayer (ML) growths from a number ofMC epithelia.The biological features andthe kinetics of cell proliferation and cell differentiation in these systems were characterised under standard culture conditions. Materials and Methods

*Abbreviations Muco-ciliary MC, Secretory otitis media SOM, Chronic otitis media COM, Monolayer ML, Amphibian phosphate-buffered saline APBS, Phosphate buffered saline PBS, Leibowitz medium L-15,Fetal calf serum FCS, Embryo extract(chick)EE, Periodic acid-Schiffs'(reagent) PAS, Alcian blue AB, Mitotic index MI, Labeled index L1, Tritiated thymidine 3H-TdR.

Differentiation 3, 79-90 (1975)

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0 by Springer-Verlag

1975

Tissue Sources 1. TheMCMembrane of theFrog's Palate. It contains a vigorous MC epitheliumwith structure similar to that of the respiratory epithelium of mammals. It is easily excised and tissue cultures can be cultivated from it at room temperature. We have used it as a model system for the investigation of mucus transport mechanism in MC epithelia [l].

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A. C. Nevo et al.:

Fig. 1. Section through epithelium of rabbit trachea, fixed immediately after excision. Pseudo-stratified arrangement ofcells. Stained AB-PAS. Different areas ofthe trachea contain different proportionsofciliated(c)cellsandofgoblet(g)cells. b-basement membrane. co1.-collagen,Mag. 5 0 0 x 3. Magnification is expressed as microscopic x photographic magnification (= 3). Mucus layer absent, washed away during the histological preparation

2. The rabbit’s trachea was chosen as the source of respiratory epitheliumof mammalian origin, as it is prone to diseases quite similar to those of man [71 and its size allows for convenient handling. 3. The MC epithelium of human adenoids. 4 . Human middle ear mucosa.

about 1 mm thick. were cut from the outer epithelial layers approximately parallel to the surface and planted as described above.

Preparation of Tissue Explants

Culture Media

I . TheMCmembrane of thefrog’s (&¶a esculenta) upper palate was excised and transferred to amphibian phosphate buffered saline(APBS)

For the culturing ofamphibian cells in vitro, investigators have usually used standard commercial synthetic media intended for mammalian cells, but diluted to reduce osmolarity. Enrichment with amphibian protein nutrients was seldom used, as amphibian cells were found to respond favorably to fluids and extracts of mammalian and chicken origin 18. 9. 10, 111. In recent years Leibovitz medium L-15(GIBCo) [I21 has beenincreasinglyused [13,14,15,16,171, supplementedwith foetal calf serum plus occasionally other nutrient sources. The osmolarity was found to be not critical,-for example, a permanent cell line of Xenopus gave high growth rates in media from 120 to 320 mOs 1151. Preliminary growth experiments were performed(-100 explants), in order to find the medium of choice, with the following media: Eagle’s minimum essential medium in Hank’s balanced salt solution.

~NaCI-O.154M(0.9%),720ml;KCI-O.15M,32ml;Phosphatebuffer M/15(Na2x IIPO,/KH2PO,),48rnl,pH-7.5;Distilled water-200mlI containing antibiotics (see Antibiotics, p. 81). Blood vessels and loose connective tissue were gently scapedoff from the inner side. Small pieces oftissue( 1-2mm’)werecut andplacedeachon acoverglassinaplastic petri dish (diam. 3.5 cm) with the collagen layer underneath, and culture medium added to a depth just enough to cover the tissue 1 mm). The cultures were kept at room temperature (between 21 and 2 4 T ) .

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2. The rabbit trachea was excised and transferred into Dulbecco’s phosphate buffered saline (PBS) containing antibiotics. It was then sliced transversally into rings 2-3mm wide, which were further cut into4 pieces (about 2 x 1.5 mm each) and the MC membrane separated from the cartilage wall with the aid of fine forceps. In order to obtain monolayer outgrowths, tissue explants were prepared on cover glasses as described for the frog. For organ culture, the explants were placed on the surface of a 0.6% agar gel containing culture medium, in small petri dishes. 3. Biopsiesfrom adenoids. removed during routine adenoidectomies, were placed immediately into PBS (containing antibiotics), cut into pieces and shaken for about a minute in a VORTEX shaker to remove excess frec lymph cells. They were rinsed in fresh PBS and thin slices,

4 . The middle ear mucosa was removed with a cupped forceps during operations, rinsed in PBS and planted on semi-solid or in liquid media.

M-199 with Earl’s modified salts. Leibovitz medium L- 15 (GIBCo). The above synthetic media were enriched with 10, I5 or 20% FCS with or without added chick embryo extract (EE) (prepared according to Richler and Yaffe [181). Final osmolarity was adjusted to 80% of mammalianmedia,withdistilled water. The best results,using as criteria extent of growth, differentiation and survival time, were obtained with L 15 medium enrichedwithFCS. Nomarked differenceswereobtained

81

Cell Proliferation and Cell Differentiation in Tissue Cultures between 10 to 20% FCS and no improvement resulted from further addition of 1% EE. MLgrowth and differentiation in synthetic media alone was either unsuccessful or very poor. The culture medium chosen for routine cultivation of frog tissue explants and MLs was: L-15-65%; FCS-15%; distilled water-20%; ph 7.4. The media were changed every 2-3 days. Respiratory epithelia of mammalianorigin were cultivated in vitro (by different investigators) in various media, mostly synthetic medium M-199 enriched with biological fluids or extracts, or M-199 alone (19, 20,21). For rabbit tracheu we performed preliminary growth experiments with M-199, L-15 and Waymouth MB 752/1 enriched with FCS (- 100 explants in each). The explants were incubated at 37’ C in an atmosphere of air with 5% CO,, except in the case ofL- 15 in which air without added CO, was used. In M-199 t FCS, viability and survival time were slightly better than in the others. No marked differences were noted at different concentrations of FCS between 5-20%. The medium chosen for routine cultivation of explants and MLs from the rabbit trachea and from human adenoids and middle ear was: M-199-90%; FCS-10%, in an atmosphere of air + 5% CO,; pH 7. I . The media were changed every 2-3 days. Semi-solidMedium. One volume of a 6% solution of agar in boiling distilled water was added to 9 volumes of the culture medium at room temperature with rapid mixing, then poured immediately into sterile plastic petri dishes and allowed to set. Antibiotics. As all the tissues used are usually contaminated at the source, bacterial growth was prevented by the use of antibiotics. The excised tissues were first transferred to APBS or PBS containing 1000 i.u.penicillin/ml, 500wg/ml kanamycinand lOOi.u./mlmycostatin.The solution was changed 3-4 times, then the explants were planted in culture media containing 100 pg/ml each of kanamycin and myocostatin and 100 i.u./ml of penicillin.

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Histochemical and Autoradiographic (AR G) Techniques Explants and MLs were fixed in Bouin’s fixative overnight, rinsed and preserved in 70% ethanol. Explants were dehydrated in graded ethanol solutions, embedded in paraffin, sectioned into 4-5 p slices and stained. Monolayers were stained directly on the coverglasses, after removal of the explants. Periodicacid-Schirs (PAS)reagent and Alcian Blue(AB). counterstained with Mayer’s hematoxylin, were used for identification of mucoid substances. PAS staining on a number of samples after diastase digestion proved thatthe positivereaction wasnot dueto the presence of glycogen. Au’toradiograrns (ARG) were prepared by dipping the slides in liquid photographic emulsion(1lfordK-S), storedin thedark for 10-12 days at 4’ C and developed with Kodak DA-19B developer. PAS stained autoradiograms wereprestained with thePAS reagent and counterstained with hematoxylin after development of the ARC. Cell nuclei synthesizing DNA were labelled by a pulse of tritiated thymidine (methyL3H)(from the Radiochemical Centre, Amersham) at concentrations of 0.5 to 1.0 pCi/ml. Microscopic Observations. The presence of beating cilia and morphological details of the cells in the monolayers in live cultures were clearly seen by phase contrast microscopy. However, due to the thickness of the tissue explants, the cilia could be clearly observed only at the periphery.

Mitotic Index(MI) was determined in the monolayers on fixed and stained preparations. TheMI obtained wasan averageover the wholeMLgrowtharound the explant. number of cells in mitosis MI = ~. total number of cells--LabeUedIndex (LI) was determined by counting thenumber of labelled nuclei in at least 500 cells. LI =

number of labelled nuclei total number of cells-

~

Results

About 1000explants each from the frog’s palate and from the rabbit’s trachea and about 600 from human adenoids were cultured. Explants from the middle ear mucosa were cultivated from only 12 biopsies. The phenotypic expressions of differentiation whose development was described were: Growth and beat of cilia,-observed in the living state by phase contrast microscopy; synthesis of mucoid substances,-determined by histochemical methods (PAS and Alcian Blue staining). Comparison of cell multiplicationrates was made from mitotic index (MI) and from labelled index (LI) measurements. The sequence ofdevelopment was qualitatively similar in cultures from the various tissue sources: cells from the epithelium near the “wound” migrated over the exposed connective tissue, and multiplied. When coming in contact with a solid substrate (glass or plastic), the cells continued to spread over it forming a coherent epithelial like monolayer sheet of cells. The new epithelial growth on the explants consisted at first of a one cell thick layer of undirerentiated (see Discussion p. 88) cells. Later, differentiationinto ciliatedcells andinto goblet cells occurred. The cells in the MLs were mostly undifferentiated to begin with and later differentiated into ciliated cells and into mucus-producing cells. Though the development of cultures from the different tissue sources was qualitatively similar, they manifested significant quantitative differences. Description of the results for each system and the differences between them follows. 1. The MC Membrane of the Frog’s Palate

a) Kinds of Phenotypic Expression of Cells in the MC Epithelium and in its Monolayer Outgrowth In the newly excised membrane of the frog’s palate, the outerlayer ofcells containscloselypackedciliatedcells and

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2a

A. C. Nevo el al.:

3

2b Fig. 2 and 3. Sections (longitudinal) through MC epithelium of frog’s palate Fig. 2a and b. Fixed immediately after excision and stained AB-PAS a. g--“goblet”; c-cilia; e-elongated cells with AB-and PAS-positive granules. Mag. 1000 x 3 b. m-densely packed cells containing AB-PAS-positive material. Mag. 500 x 3 Fig. 3. New epithelial growth at edge ofexplant: 4 daysin culture. Stained PAS + AB. Mag. 500 x 3. Note continuous row ofciliated cells (c) with few goblet cells (8)

83

Cell Proliferation and Cell Differentiation in Tissue Cultures

mucus producing cells. Among the latter are mainly two differently appearing types: elongated granulated cells which stain intehely with PAS and AB, and cells with distended “globlets” whose contents stain less intensely with PAS and very weakly with AB, and whose circumference contains material which stains strongly AB-positive and PAS-positive (Fig. 2a). Occasionally a third morphological type of AB-PASpositive cell is observed (Fig. 2b). The cells are densely packed, forming a continuous outer layer with no ciliated cells, reminiscent of a gland. During differentiation of new epithelialgrowth, elongated granular cells appear first and the goblets later. In the MLs a number of morphologically different cell types are also observed: 1) Flattened cells which are “undifferentiated”,--do not contain cilia and do not stain either PAS or AB-positive. A round or oval nucleus containing one or more nucleoli is clearly seen in these cells by phase contrast in the live state. 2) Cells with cilia. 3) Cells containing pear-shaped clusters of granules which usually stain both AB- and PAS-positive, but may sometimes stain only AB-positive and PAS-negative (Fig. 5). 4) Cells containing a spherical “goblet”, whose contents

stain predominantly PAS+ and weakly or negatively to AB and whose circumference contains strongly staining AB and PAS positive material (Fig. 5).

b. Development of the Monolayers The sequence of events is divided into stages, for convenience of description: Stage A ML growth appeared around the explants within 1-2 days after planting, consisting entirely, or mostly, of undifferentiated cells. The MLs formed coherent epithelial-like sheets of polygonal cells. The leading edge showed ruffled membranes. Duration - 2-4 days in culture, from time of planting. Stage B Cells in the ML differentiate into ciliated cells and into mucus producing cells (AB/PAS-positive) (Fig. 4). The cilia grow upwards and begin to beat, at first in an uncoordinated manner; but as they attain their full growth, a metachronic beat develops in each cell. Ciliated cells develop more or less randomly in the ML and the direction of the metachronic wave in each cell is unrelated to that in other cells. In the sequence of development of the AB-PAS-positive cells, the clusters of granules appear first and the “globlet” develops later.

The ML still forms a whole coherent sheet of flattened cells. Duration of stage B-up to 6-8 days in culture. Stage C Cells in the ML, in certain areas, become closer packed. In spots they recede and form “holes” in the ML surrounded by thickened growth resembling an active ciliary epithelium. Usually, a degree of coordination of the metachronic wave develops in such growths, forming a continuous wave. Duration-3-4 weeks. Stage D Cells degenerate and detach from the sub strate. Degeneration and disappearance of the ML occurs within a few days. One must bear in mind that in stage B relatively large areas,mainlyintheperiphery,may stillappear asinstageA (Fig. 4), and in stage C theremay still be areas appearing as in stage B or even as in stage A. Both in the Explants and in the MLs, ciliated cells and mucus-producing cells did not develop at the samerate and did not maintain a constant ratio. In many cases ciliated cells developed first and later the ratio of goblet cells to ciliated cells increased. However, the sequence was sometimes reversed. c. Growth of the Monolayers with and without Explants The explants were removed after different durations of ML growth and the MLs were cultured further until the end of growth and degeneration (stage D). The results are represented in Table 1. It will be seen that the isolated MLs remained viable about as long as the controls (in which the explants were not removed) and underwent the same sequence of development. When the removed explants were replanted, new ML outgrowths occurred, which went through the same stages of development from A to D.

The Muco-ciliary Membrane of the Rabbit Trachea Experiments were performed with ca 650 explants on semi-solid media and 350 explants in liquid media. The explants were usually cultured for periods up to 15 days, during which they remained viable and showed vigorous ciliary activity. Monolayer outgrowths began 2-4 days after planting, from most of the explants. Ciliated cells appeared almost from the beginning but as the MLs grew they remained confined to the area around the explant, to a distance of 1-1.5 mm. The number of mucus-synthesising (PAS’) cells in the MLs was very small and they were found only near the explants. The cells in the periphery were large, more loosely packed, of irregular shape and often contained multiple nuclei. The width of the MLs usually did not

84

4

6

A. C. Nevo et al.:

5

7

Cell Proliferation and Cell Differentiation in Tissue Cultures

85

Table 1. Comparison of monolayer growths from MC epithelium of frog’s palate with and without explant

Experi ment

No. of sample

I 8

I1

I11

IV

9 10 11 12 13

Removal of explant after days

Stage of ML at time of removal

End of Stage C days

End of Stage C of control days

10 10 10 10 10 10 10

C(+B) C(+B) C(+B) C(+B) C(+B) C(+B) C(+B)

23 29

26 25 25

14 14 14

25 30 24 28 28 35 31

36 33 32

35 32

35

14 15 16 17 18 19 20

18 20 20

C C C C C C C

21 22

25 25

C (advanced) C (advanced)

18

Figs. 4 and 5. ML outgrowth from frog’s palate MC membrane. Fig. 4. Photomicrography oflive culture. StageB, 5 days after planting. Many cells with granular clusters (arrows), and among them ciliated cells (cilia not seen in photograph). Undifferentiated cells towards periphery. Mag. 200 x 3 Fig. 5. Stage B, 6 days in culture. Stained AB-PAS. Goblets(g) contain predominantly PAS+material surrounded by granular material staining predominantly AB+ tinged with PAS+ (violet-bluecolor): pear shaped granular clusters stain predominantly AB+, ranging from blue (pure ABt) (1) to blue-violet (2). Mag. 1000 x 3

Fig. 6. Section from explant of rabbit trachea. 4 days in culture; 24 h pulse of3H-TdR on last day before fixation. Arrows showlabellednuclei of proliferating stem cells penetrating the connective tissue (epithelial “cord”). Mag. 500 x 3 Fig. 7. ML from the frog’s palate MC epithelium. Cell in anaphase containing PAS’ material (m). Mag. 1000 x 3

29 29 24 24 23

exceed 3-4 mm, which they attained after 12-16 days in culture. After 2-3 weeks in culture, cells at the periphery degenerated, while close to the explants they remained viable with continuing ciliary beating. Culturing for periods of over 3 weeks was usually difficult because of rnultiplication of fibroblasts or of bacterial growth (apparently from original contamination of the tissue source). In medium containing a 1 : 1 mixture of M-199 and L- 15 (enriched with FCS as usual) development during the first 6-8 days was similar to that described above and in Table 2. But then, in about 50% of the cases (44 explants), theMLs started to grow rapidly, covering the whole areaof the coverglass (18x18 mm) after 7-8 days of further growth. The cellsdid not differentiate and formed a characteristic epithelial-like ML. LI was very high, approaching 100%oflabellednucleiafterapulseof24h, especially atthe periphery.

A. C. Nevo et al.:

86

Table 2. Comparison between the development of MLs from the MC epithelium of the frog’s palate and of the rabbit’s trachea

Frog’s Palate

Rabbit’s Trachea

1. MLs were viable for 4-5 weeks. Expansion of the MLs ceased after ‘8- 12 days.

1. MLs were viable for 2-3 weeks.

2. Differentiation occurs throughout the ML, including the penphery.

2. Differentiation confined to area near the explant.

3. P A S cells and ciliated cells are numerous: Differentation proceeds to almost complete transformation of the stem cells, resulting in growths resembling an active MC epithelium.

3. P A S cells very few, though many ciliated cells. Closely packed epithelial-like MC growths did not form.

4. In cultures 1 week old or more, mitotocally active cells were found in undifferentiated areas and not found in densely differentiated areas.

4. Mitotically active cells in 1-2 week old cultures were distributed

5. AFter removal ofthe explants, theMLs underwent the same sequence

5. After removal of the explants, the MLs degenerated within a few

of development and of approximately the same duration as those in contact with the explants.

Differences between the development ofMLs from the MC epithelium of the rabbit’s trachea and of the frog’s palate are summarised in Table 2. In explantsfrom the rabbit trachea, the MC epithelium showed a pseudostratified arrangement with varying density of goblet cells (Fig. 1). Submucosal glands were very few and found mainly in the proximal part. During the first 5-7 days in culture on semi-solid medium the epithelium maintained its normal structure, after which it gradually lost its pseudostratification, becoming stratified and in many cases showing hyperplasia ofthe basallayer. Ciliated cells with active ciliary beat and goblet cells persisted throughout during the whole period of cultivation ( 1 2- 15 days), but the amount of PAS+ material diminished. After 7-9 days in culture the connective tissue degenerated quite rapidly and consisted of a loosely packed collagenous network with only a few viable cells. The basal membrane often broke down and the connective tissue was frequently penetrated by cords or massesof rapidly proliferating basal cells (Fig. 6). Vacuolisation appeared in many cells after 10 days in culture. After 5-6 days in culture, about 50% of the explants were almost completely encircled by new epithelial growth, which covered the denuded connective tissue. The new epithelium was mostly bilayered, with the outer layer consisting mainly of undifferentiated cells and some ciliated and PAS+ cells. The changes of the epithelial structure of explants cultivated in liquid medium were similar to those described above, but stratification appeared earlier (3-4 days).

Expansion of the MLs ceased after 8- 10 days.

mainly near the explant.

days.

3. The MC Epithelium of Adenoid Tissue

Explants on semi-solid medium and in liquid medium containing M- 199 were maintained in culture up to 15 days during which they remained viable and showed ciliary activity in about 50% of the cases. M L growths began 4-7 days after planting (in about 60% of the explants). In general, the number of cells which underwent differentiation was relativelyfew andthesewere mostly ciliated cells. Ciliary beat was maintained during 4-8 days of ML growth. On semi-solidmedium the new epithelialgrowthcovered the exposed surface of the explants almost completely after 5-8 days in culture. The original epithelium of the adenoids is not uniform in structure, containing sections that are pseudostratified, stratified, or of a transitionaltype. PAS+ cells are relativelyfew. After a few days culture.most of the epithelium became stratified, with fewer PAS+ cells; cords of basal epithelial cells penetrated into the disintegrating lymphatic tissue. 4. The Middle E a r Mucosa

Explantswerecultivatedfromonly 10biopsies onsemi-solid medium, one from a healthy person and 9 from patients suffering from serous otitis media and from COM. Explants were almost completely covered with new epithelial growthafter 3-5 days whichwasmono-orbi-layered, with few ciliated cells and very few P A S cells. Explants from 2 additional biopsies were cultivated in liquid media and ML outgrowths obtained similar to those grown from the adenoid epithelium.

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Cell Proliferation and Cell Differentiation in Tissue Cultures

5.Expansion ofEpitheliaEandMonolayerGrowths andthe Distribution of Mitotically Active Cells

a.In theMCepitheliurn ofexplantedtissues in vitro, as well as in vivo,when using 3H-TdRas a DNA precursor only the basal cells acquiredthe label and werethereforeundergoing the cell division cycle. The ciliated cells and mucusproducing cells did not divide. As the new epithelial growths consisted at first of a single layer of undifferentiated cells, new cells could be fed into the expanding growths only by multiplication and/or migration of basal type (undifferentiated) cells. Our experiments showed further that mitoti-

migration of stem cells from the area of the “wound” before their multiplication. Increasedmitotic activity in theepithelium near the wound and in the epithelial growth was observed after about a day in culturein the case ofthe frog’s palate epitheliumand after 2-3 days in culturein the case of the rabbit’s trachea, reaching a maximum after -6 days (Fig. lo). In some explants a LI approaching 100% from a

700

500 2

+

300

u

days

07

100

Fig. 9. Labelled index (LI)in ML growth from rabbit tracheaepithelium asafunctonofdaysinculture.(- - - - -) 199;(----)L-l5,Thefigures give the number of explantsexamined. 24 h pulseof0.5 uCilM1’H-TdR before fixation.

05

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l

1

1

1

l

T

103-

i

02 01

005

0

0

2

1

4

6

’

8

.

1

pf’

0

days in culture C

20 I

O2

7

*-$‘ ! ’ 2 b

I

2

3 days

cally active cells were distributed throughout the expanding epithelium and ML,-not only near the ”wound” or in the advancing edge of the epithelial sheet, to mention the extreme possibilities. New growth on the denuded connective tissue of the explants appeared already after 5-6 hin culture in the case ofthe frog’s palate epithelium, and within 24 hin the case of the rabbit’s trachea. Autoradiograms of sections of the explants, which were exposed to 3H-TdR from the time of introduction into the growth medium to the time offixation, showed that at this early stage the cells were mostly unlabelled. Thus the early epithelial growth occurred by

in

2

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4

5

-

~

6

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7

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Fig. 10. Llinexplantsfromrabbittracheaduring 1st weekofcultureon semi-solid medium 199 + 10% FCS. 24 h pulseof 0.5 pCi/ml 3H-TdR beforefixation. LIwas calculated as % oflabelled nucleipertotalnumber of basal cells. (0)LIin theoriginalepithelium(atadistance > 1000pmfromtheedge. -200 cells counted per measurement. (e)-LT in the epithelium near the wound (to a distance of 700 120 cells) and in the new growth. Figures on the graphs represent number of explants examined.

24 h pulse was obtained. In the original epithelium (at a distance from the wound) the LI remained constant. Continuous exposure to 3H-TdRfrom time of planting up to 93 h in culture, of both frog and rabbit tissue explants, did not result in MLs with LI of 100%. Among the unlabelled cells were also some differentiated cells. It may be concluded, therefore, that stem cells can migrate compa-

88 ratively large distances and then differentiate without cell division immediately preceding differentiation.

b. Expansion of the monolayers continued up to about 8 days in culture, sometimes longer, after which they ceased to grow outwards. The maximum distance from the explant to the edge of the ML was usually 2.5-3.0 mm (Fig. 8a). In MLs from the frog’s tissue, the MI during the first days of growth (in stage A) was high (4-5%) then declined rapidly in stage B and reached very low values in stage C (Figs. 8b and c). During stage A, pulse-labelling with 3H-TdRshowed that labelled nuclei were distributed fairly evenly throughout the MLs, but in advanced stage B and in stage C, after extensive differentiation, labelled nuclei were found almost exclusively in the undifferentiated areas, mainly towards the periphery. However, careful observation of PAS-stained preparations revealed occasional PAS+-stained cells which contained labelled nuclei or were undergoing mitosis (Fig. 7). Cases of ciliated cells undergoing mitosis in live cultures were also observed, with the cilia continuing to beat during cytokinesis. Thus loss of mitotic activity in differentiated cells in theMLs was not complete as in the tissue framework. In MLs from the rabbit trachea after 1-2 weeks in culture, mitotically active cells were found mainly near the explants, in areas containing also differentiated cells. The decline in the overall mitotic activity as a function of time in culture is shown in Fig. 9. Mitotic activity in medium containing L- 15 was significantly higher than in medium containing M-199 during the first days of ML growth.

6. Cell Movement and Migration within the MLs Though the MLs form coherent sheets ofcontiguous cells, observation of live ML cultures, by direct observation with the phase contrast microscope or by time lapse cinematography, showed that cells move about and change position in the ML. In order to find out whether they can move about to the extent of changing neighbours (and hence severing all former cell junctions), explants were given a short pulse of 3H-TdR(3h)at planting andthen cultured4-5 days,until a good ML growth was obtained, and autoradiograms prepared. It was found that many labelled cells were surrounded completely by unlabelled cells, often to a distance of many cells. Since the label was acquired by basal cells in the explant and then passed on equally to their daughter cells, this observation proves that they changed neighbours and relative positions during ML growth.

A. C. Nevo et al.:

Discussion

From the viewpoint of embryology, stem cells of adult (post-embryonic) tissues are differentiated in the sense that they are committed to produce certain kinds of specialised cells. In describing our results, however, it has been convenient to apply the term undzgerentiated to stem cells and to cells in the MLs in which the specialisedfeatures characteristic of the tissue are absent, i.e., in which the epigenotype (the heritable ability to differentiate into a particular cell type) is unexpressed. In the following we consider some aspects of proliferation and cytodifferentiation in our experimental systems.

I . The Phenotypic Expression in our Model Systems has not been Altered by theArtiJica1Conditions of Cultivation in vitro. Our results show that cells growing in a monolayer sheet, outside the organised tissue framework, retained their characteristic developmental traits, dividing and differentiating into ciliated and into mucus-producing cells. Hence the epigenotype has not been altered or de-differentiated. Are the AB- and PAS-positive granulated cells and goblet cellsintheepithelium andintheMLdifferent types of terminally differentiated cells, or stages of development of one type of cell-the goblet cell? Observations tending to support the former supposition are: (a.) From histoligical sections it appears as if both kinds of cells are secreting. (b.) Smears of secreted mucus stain inhomogeneously,-mostly PAS+ material interspersed with PAS- and AB-positive material. On the other hand, there are observations supporting the latter supposition: Both in the new epithelium and in the ML, the granulated PAS- and AB-positive cells appear first and the fully developed goblet cells later. The goblet cells also contain around the goblet AB- and PAS-positive granules suggesting a sequence of development from one kind to the other. Perhaps the normal development of the goblet cell proceeds from the stage of the granulated cell to that of the swollen goblet cell, which secretes predominantly PASt material, while some cells may secrete when still in the pre-goblet stage, adding ABt materialto themucus blanket here and there.

2. GrowthofNew EpitheliumandExpansionoftheML Occur by Proliferation and Migration of the Stem Cells Throughout the New Growths. Histochemistry, morphology and DNA labelling showed that undifferentiated (stem) cells migrated from the area of the wound over the exposed connective tissue and continued to spread over the solid substrate. Cell division and differentiation took place throughout the new epithelial and ML growths. Thus

Cell Proliferation and Cell Differentiation in Tissue Cultures

expansion ofthe MLs occurred by continuing proliferation of the stem cell progeny. The MLs ceased to expand due to ( 1 ) reduction in MI values, (2) change in the surface properties of the cells, (3) degeneration of the cells. In the MLs from the frog’s cells, it will be seen (Fig. 8) that they cease to expand at the onset of stage C and this coincides more or less with a sharp decline ofthe MI to verylow values. Thedecline inthe (average) MI can thus be explained by the increase in the proportion of differentiated cells, which in general do not divide. However, there also occur areas of undifferentiated cells which are not proliferating. They could be in the resting stage (Go).Altered surface properties of the cells in stage C also play a part, as the cells cease to extend themselves and tend to round up and even recede. In the case of MLs from the rabbit trachea and from human adenoids, loss ofmitotic activity occurredmainly in the peripheral areas containing undifferentiated cells. The morphology ofthe cells suggeststhatthey mayrepresent an early stage of cell deterioration, thus limiting the expansion of the ML.

3. The Regulatory Mechanisms were not Effective in M L Growth Outside the Tissue Framework. In MLs from the MC membrane of the frog’s palate, transformation of the stem cells was almost complete, leaving no stem cell compartment from which renewal of the system could occur. In contrast, in the MLs from the rabbit trachea and from human adenoids and middle ear mucosa, the proportion of cells which underwent differentiation was smaller, with relatively few PAS+ cells, and differentiation was confined to the vicinity of the explant. It seems reasonable to assume that loss of control of proliferation and differentiation in the MLs is due to absence ofthe particular multicellular patterns of relationships between the cells in the tissue, upon which presumably such control depends. 4 . Cell Division and Cyto-differentiation were not always Mutually Exlusive. It has been reported that in stratified squamous epithelia (keratinising) [221 and in respiratory epithelia [231 only basal cells proliferate. Terminally differentiated cells of the surface layer did not divide. Blenkinsopp [241, on the other hand, found that in the rat trachea 1.3% ofthe superficial cells were synthesising DNA while containing P A S material. We found that in explanted MC tissues in vitro, differentiated cells in the surface layer did not divide, but in the MLs both ciliated cells and mucus producing cells did occasionally divide. According to Holtzer and Abbott [251 “the distinction between a terminally differentiated cell and its precursor cell lies in the unique sets of luxury molecules produced by

89 each”. They proposed that “synthesis of luxury molecules . . .is governed by an all or none type ofregulatory system”. In most kinds of cells, initiation of the synthesis of luxury moleculesis accompanied by blocking DNA synthesis and mitosis, but this is apparently not necessarily SO. Thus Cahn and Lasher [26] demonstrated that chondrocyte clones which were synthesising DNA were also synthesising sulfated cartilage matrix, and Davies et al. [271 showed similarly in fibroblast cell cultures that synthesis of collagen and ofDNA were not mutually exclusive. Goldstein et al. [28] showed that neonatal cardiac cells of 2-day-old rats containing well-aligned myofibrils were also synthesising DNA. 5. Cell Proliferation and Cell Digerentiation in the M L s were Affected by Proximity of or Contact with the Explant. In the case of the rabbit trachea, the fact that cell proliferation and cell differentiation in the MLs were usually confined to the vicinity of the explant and that the MLs deteriorated rapidly after its removal indicate that presence ofthe tissue helps preserve the viability ofthe cells in the ML and may also affect their differentiation. Whetherthese effects are dueto diffusiblefactors manufactured by the tissue or whether they are mediated through cell-to-cell contact has not yet been established. By contrast, frog cell monolayers were not as dependent on the presence of the explant,-they underwent the same sequence of differentiation without the explant as when in contact with it, though they did not proliferate to the same extent.

6. Conditons Inducing Rapid Cell Division. The extraordinary stimulation of cell division in MLs from the rabbit trachea by enriched mixture of L-15 and M-19 merits some comment, though the investigation of this problem has not been pursued beyond the preliminary stage. The following facts should be noted: 1)Rapid proliferation without differentiation occurred in the peripheral area and continued also after removal of the explant. 2) The change occurred after about a week’s growth, during which the usual developmental features were manifested. 3 ) The rapid expansion of the MLs was due to a mass transformation of the cells, since it occurred around the whole circumference of the ML. The nature of this change remains to be elucidated.

7. Does the Stern Cell Population in MC Epithelia Consist of a Single or of Several Epigenotypes? Stem cells of MC epithelia are morphologically and histochemically all alike, though they normally develop into two distinct phenotypes,-ciliated cells (C) and mucus-producing cells

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(M). If each phenotype is the expression of a different epigenotype then the question before the cell is “to differentiate or not to differentiate”; on the other hand, if the phenotypes are rnoduEations (in the terminology of P. Weiss [291) of a single epigenotype, the cell must also “choose” between alternative developmental pathways in response to environmental cues. That a stem-cell population could be induced by a chemical signalto switch from one type of differentiation to another has been shown by Weiss [291 in the case of mucous metaplasia produced by vitamin A in embryonic chick epidermal cells. Epidermal cell suspensions (dispersed by trypsin treatment) after brief contact with the chemical transformed into an actively secreting mucous epithelium. Fell andMellanby [301showedthat the vitamin A induced mucous metaplasia in explants of chick embryonic epidermis was reversible and Fitton-Jackson and Fell 1311 reported that “the same cell often contained both secretory globules and prekeratinous filaments”. Though these experiments do not yet prove that in normal MC epithelium homeostatic mechanisms control the dgerentiation of an equi-potent stem cellpopulation,it would seem plausible. Our observations that in new epithelial and MLgrowths differentiation to M cells and to C cells does not proceed at the same rate or according to a fixed time sequence eliminates the possibility that the usual course of differentiation is by one daughter cell from a quanta1 (the mitosis preceding differentiation) mitosis differentiating into a C cell and the other into an M cell, as this would require that M and C cells be producedin a 1 : 1ratio in adjacent pairs. However, this does not give an unequivocal answertothequestion ofoneor several epigenotypes. A definite answer could beobtained in principleby cloning the stem cells, but so far our attempts at cloning have not been successful. Conclusion The in vitro experimental systems derived from different MC epithelia have many developmental characteristics in common and should be suitable models for the investigation of cell proliferation and cell differentiation in these tissues. Because oftheir simple structure, they should prove of great value in elucidating the control mechanisms in post-ernbrvonic epithelial tissues.

Acknowledgements, This investigation was supported in part.by Grant No. Ns 10048 from the National Institutes of Health. The authours wish to thank Rina Levi and Ilina Drucker for their devoted technical assistance.

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