Cell Tiss. Res. 178, 333-339 (1977)

Cell and Tissue Research (~', by Springer-Verlag 1977

DNA-Synthesizing Cells in Human Fetal Thymus C. R6pke, B. van Deurs, and P.E. Hoyer Anatomy Department A, University of Copenhagen, Denmark

Summary. Fragments and suspensions of human fetal thymus were incubated in the presence of 3H-TdR to permit study of the distribution and morphology of DNA-synthesizing cells. Results of light and EM autoradiography showed that 1. although DNA-synthesizing cells were present in the medulla, the vast majority of these cells were localized in the thymic cortex, 2. cells with the typical EM appearance of small lymphocytes and lymphoid blast cells both synthesized DNA, and 3. cells in S-phase were predominantly 8 to 12 ~tm in size.

Key words: Thymus, Human fetus - Lymphocytes - DNA synthesis Autoradiography - Electron microscopy. Introduction It is generally acknowledged that two populations of lymphocytes-thymusderived and bone marrow-derived ones-exist in peripheral tissues of man (Raft, 1973). Although numerous experiments have established these functional categories of cells with respect to mainly peripheral blood lymphocytes, our knowledge of the basic kinetics of lymphoid cells is still based on findings in rodents (Osmond, 1972). This is especially true of the cell kinetics of the primary lymphoid organ of cellular immunity: the thymus. Kinetics of thymocytes have been studied intensively in rodents (surveys: Everett and Tyler, 1967; Osmond, 1972), b u t - t o our knowledge-only one report has dealt with kinetics in the human thymus (Papiernik, 1972). We therefore found it well-warranted to study the cell populations in this organ. Knowledge of kinetics of cells in the fetal thymus is relevant, since this organ may be used in treatment of patients with dysfunction of the cellular immune system (Cleveland et al., 1968; Wara et al., 1974) and kinetic information is needed for a further understanding of the ontogeny of immunity in man. Send offprint requests to: Dr. C, R6pke, Anatomy Department A, 71, Raadmandsgade, University

of Copenhagen, DK-2200 Copenhagen N, Denmark

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Furthermore, fetal thymuses are obtainable under optimal conditions from l e g a l a b o r t i o n s , a n d it h a s b e e n s h o w n t h a t f e t a l t h y m o c y t e s a r e a b l e t o f u n c t i o n l i k e m a t u r e cells i n s e v e r a l i m m u n o l o g i c a l t e s t s , a s r e v i e w e d b y S t i t e s et al. (1975).

The present experiments were set up to characterize the morphology of the cells which synthesize DNA in the human thymus, and the distribution of these cells in the thymic cell populations. In addition, the distribution of cells in DNA synthesis within the thymus was evaluated.

Materials and Methods Human fetuses were obtained at the time of hysterotomy performed for therapeutic abortion. Of these, two fetuses aged about 16 weeks (CRL: 144mm and 151 mm, both females) were selected for the present study. The thymus was removed within 1 h after hysterotomy. Cell suspensions were prepared immediately after removal of the thymus. The thymus was cut in fragments with razor blades. Some fragments were used for incubation with 3H-Thymidine (3H~ or processed directly for EM. The remaining fragments were minced with scissors in RPMI 1640 medium containing 5 ~ calf serum. After filtration of the resulting cell suspension, the concentration was adjusted to 107 cells per ml. Two I~Ci 3H-TdR (spec. act.: 6.7 Ci/mmol) was added per ml cell suspension, and the suspensions were incubated at 37~ in an atmosphere containing 5 ~ CO 2. The suspensions and the tissue fragments were incubated for 45 or 90 min. Thereafter cold medium was added, and the cells or tissue fragments were centrifuged and washed 3 times at 4 ~C. Some suspensions were used for smears and coated with Kodak NTB2 emulsion for autoradiography; exposure time: 2 days. Other suspensions and the tissue fragments were processed for 1 lain thick sections and EM: they were fixed for 1 h in 0.1 M sodium cacodylate buffer, pH 7,2, containing 2.5~ glutaraldehyde. The same buffer containing 1~ OsO4 was used for postfixation before embedding in Epon. 1 ~tm thick sections were processed for autoradiography in the same way as the smears and were developed after 3, 4 or 6 weeks. For electron-microscopical autoradiography, silver to gold sections on copper grids were covered with Ilford L4 emulsion. After exposure for several months, the autoradiograms were developed in Kodak D 19, and contrasted with uranyl acetate and lead citrate. Lymphoid cells were classified in smears by cell diameter: cells with a diameter less than 8 ~tm were counted as small lymphocytes, medium lymphocytes included cells with a diameter of 8 to 12 ~tm and large lymphocytes had a diameter of more than 12 ~tm (up to 20 I~m). In 1 ~tm sections and in EM photos, cells were classifed according to nuclear diameter and density as well as cytoplasmatic morphology (see results). One thousand or more cells from each category were counted in smears for evaluation of the percentage of labeled cells. Based on grain distribution over 1000 erythrocytes, cells with 3 or more overlying silver grains were included in the labeled group. To evaluate the distribution of small, medium and large thymocytes within the thymic lobes, photographs were taken of representative parts of the superficial cortex, the deep cortex and the medulla. Twenty equally sized areas (0.013 mm2) were selected from the photographs of each of the 3 parts of the lobes and the cells counted. Numbers and percentages in the text and the table are given as the mean + standard error of the mean.

Results S m e a r s o f s i n g l e cell s u s p e n s i o n s w e r e u s e d f o r e v a l u a t i o n o f t h e p e r c e n t a g e s o f s m a l l ( u p t o 8lzm), m e d i u m s i z e d ( 8 - 1 2 ~ t m ) , a n d l a r g e t h y m o c y t e s ( m o r e t h a n 12 Ixm). I t w a s f o u n d t h a t 74.4 + 3 . 9 ~ o f t h e cells h a d t h e t y p i c a l a p p e a r a n c e o f

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Fig. 1.3H-TdR labeled cells from an autoradiographic smear of a suspension of fetal thymus, incubated in vitro for 45 min in the presence of 3H-TdR. From the left to the right: one large, two medium-sized blast cells, and one small lymphocyte. Fetal age: 16wks. x 1000

small thymocytes: scanty cytoplasm and a round nucleus with condensed chromatin. Medium-sized thymocytes constituted about 22.2 + 1.5~ of the cells and showed generally 1-2 nucleoli in addition to more lightly stained nuclei than those of small thymocytes. The cytoplasm was more abundant and generally more basophilic in medium-sized thymocytes than in small thymocytes. The remaining 3.4---0.8~ of the cells-the large thymocytes-had the typical appearance of blastoid cells. The nuclei exhibited finely dispersed chromatin except for some membrane-associated condensations. One or more nucleoli were usually present. Autoradiographic results obtained from these smears showed that 1.7 +0.5% of the small thymocytes had incorporated 3H-TdR during the incubating period. Further it was found that 56.3 + 3.2~o of the medium-sized thymocytes and 45.0 + 2.7~o of the large thymocytes were labeled i.e. had been synthesizing DNA. No significant differences were found between suspensions which had been incubated for 45 min and those which had been incubated for 90 min in the presence of 3H-TdR. Light-microscopic observation of autoradiographs of 1 Bm thick sections revealed that the vast majority of labeled cells were situated in the cortical area close to the capsule (Fig. 2a). However, this finding was partly due to limited penetration of 3H-TdR into the thymic lobes. In lobes which were cut through the medulla or the deep part of the cortex, labeled cells were found in both these areas, the finding of DNA-synthesizing cells in the medulla being of particular interest (Fig. 2b). By combining the autoradiographic results obtained from smears with cell counts and estimation of cell size in the superficial and deep cortex and the medulla (Table) it was, however, apparent that the cortex was the site where the vast majority of the DNA-synthesizing cells are to be found. To further investigate the morphology of thymic cells engaged in DNAsynthesis, Epon-embedded suspensions and fragments of the fetal thymic lobes were prepared for EM autoradiography. In general the ultrastructural preservation of the thymic cells was reasonable, in particular when the long incubation time in the medium before fixation is considered. The chromatin, however, sometimes exhibited some "attenuation", appearing gray rather than dark in the EM. As indicated by the smears and 1 Bm thick sections, some small thymocytes were found to be labeled. These cells were characterized by their scanty cytoplasm with free ribosomes. Only a few organelles (mitochondria and small Golgi complexes) were present. The mostly round nuclei were dark (due to condensed

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Fig. 2. A Autoradiograph of the superficial cortex of a thymic lobe from a 16 wk old fetus. Note that 3H-TdR labeling is predominantly present in the larger lightly-stained cells. Section thickness 1 ~tm. • 790. B Autoradiograph of the medulla of a thymic lobe from the same fetus. Note the Hassall's corpuscle and labeled cells of various sizes (arrows). Thickness of section 1 ~tm. x 600

Table 1. Amounts of small, medium sized and large thymocytes in equally sized areas (0.013 mm 2) of superficial cortex, deep cortex and medulla of fetal thymic lobes

Superficial cortex Deep cortex Medulla

Small

Medium

Large

Total

35.6 • 2.2 54.1 +3.6 24.4 -+ 1.3

25.6 • 3.7 17.8 -+ 1.6 9.3 •

8.4 • 0.9 2.9 • 1.0 1.5 -+0.3

68 75 34

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Fig. 3 A - C . E M autoradiographs o f cells from a suspension made of t h y m u s from a 16 wk old fetus. The cells were incubated for 45 min in vitro in the presence of 3H-TdR. A Labeled small thymocyte, B and C Labeled cells with blast cell appearance. • 8400

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chromatin). Labeled larger lymphocytes (blasts) exhibited relatively more cytoplasm, often with several mitochondria, and well-developed Golgi complexes. The nuclei were often irregular in shape with peripheral chromatin condensations and appeared lighter than those of small thymocytes. Based on EM photos it was not possible to distinguish between a well-defined group of "medium" lymphocytes and larger "blastoid" cells.

Discussion

In the present study the vast majority of labeled cells of the human thymus had the appearance of lymphoid blast cells as found by light and EM autoradiography. However, some typical small thymocytes showed DNA synthesis. This latter finding is of particular interest since it is generally accepted that small thymocytes and lymphocytes do not synthesize DNA in the absence of stimulation (e.g. antigenic), thus these cells generally do not contain tracer the first 2 h after injection of 3H-TdR into rodents (Everett and Tyler, 1967; Osmond, 1972; R6pke and Everett, 1975). However, it may be that the small cells found to be labeled in the present study represent the extreme tail of the size distribution curve of the population of DNA-synthesizing medium and large thymocytes, as suggested by Osmond (1973), although the EM morphology indicates that these cells belong to the population of small thymocytes. It is significant that the labeling percentage of medium-sized thymocytes was even higher than that of large cells of the present study. Comparable results were obtained by Papiernik (1972). When the percentage distribution according to cell size and the labeling percentages of the small, medium and large cells are combined it is seen that labeled medium-sized cells constitute 12.5% of all thymic cells, a high number compared to the 1.3 and 1.5~o labeled large and small thymocytes. From the labeling percentages and the morphology of the labeled cells it is not possible to evaluate whether medium-sized thymocytes and large thymocytes are included in the same population or whether they represent two generations of cells, the larger giving rise to the medium-sized thymocytes as suggested by findings in rodents (Sainte-Marie and Leblond, 1964; Hinrichsen, 1965; Borum, 1968). We must conclude from the present study that the majority of thymocytes with diameters from 8 to 20 ~tm share nuclear and cytoplasmatic morphology and are in a rapid cell cycle. Furthermore, the present study is the first to evaluate the localization of DNA-synthesizing cells in the lobes of the human thymus. Both labeling percentages and distribution of the different cell types (according to cell size) within the thymic lobes indicate that the cortex is the major production site of thymocytes, as is true of rodents (Sainte-Marie and Leblond, 1964; Hinrichsen, 1965; Borum, 1968). However, DNA-synthesizing thymocytes of all sizes are found in the medulla (Fig. 2b), which is in agreement with results obtained in mice (Potmesil and Goldfeder, 1973). As can be seen from the figure published in the present paper, typical Hassall's corpuscles were present in the medulla of the thymic lobes. This may indicate that thymuses from this period of prenatal age are

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morphologically mature, and that the present results may also be valid for the post-natal thymus. Immunological studies have indicated that thymocytes from

fetuses of this prenatal age are functionally mature (Stites et al., 1975). Finally we wish to stress that it is possible to use human fetal tissue for culture and that its morphology looks reasonable as judged by EM, even after incubation at 37 ~ C for 90 min.

References Borum, K.: Pattern of cell production and cell migration in mouse thymus studied by autoradiography. Stand. J. Haemat. 5, 339-352 (1968) Cleveland, W.W., Fogel, B.J., Brown, W.T., Kay, H.E.M.: Foetal thymic transplant in a case of diGeorge's syndrome. Lancet 196811, 1211 1214 Everett, N.B., Tyler, R.W.: Lymphopoiesis in the thymus and other tissues: Functional implications. Int. Rev. Cytol. 22, 205-237 (1967) Hinrichsen, K.: Zellteilungen und Zellwanderungen im Thymus der erwachsenen Maus. Z. Zellforsch. 68, 427 444 (1965) Osmond, D.G. : The origins, lifespans and circulation of lymphocytes. In: Proc. of the Sixth Leucocyte Cult. Conf. Edit. by M.R. Schwarz, pp. 3-32. New York: Academic Press, Inc. 1970 Osmond, D.G., Miller, S.C., Yoshida, Y.: Kinetic and haemopoietic properties of lymphoid cells in the bone marrow. In: Ciba Found. Symp. 13. Edit. by J.F. Loutit, pp. 131-156. Amsterdam: Ass. Scient. Publ. (1973) Papiernik, M.: Ontogeny of the human lymphoid system: Study of the cytological maturation and the incorporation of tritiated thymidine and uridine in the foetal thymus and lymph node and in the infantile thymus. J. cell. Physiol. 80, 235-242 (1972) Potmesil, M., Goldfeder, A.: Nucleolar morphology and cell proliferation kinetics of thymic lymphocytes. Exp. Cell Res. 77, 31-40 (1973) Raft, M.C.: T and B lymphocytes and immune responses. Nature (Lond.) 242, 19-23 (1973) R6pke, C., Everett, N.B.: Life span of small lymphocytes in the thymolymphatic tissues of normal and thymus-deprived BALB/C mice. Anat. Rec. 183, 83-94 (1975) Sainte-Marie, G., Leblond, C.P.: Thymus-cell population dynamics. In: The thymus in immunobiology. Edit. by R.A. Good, A.E. Gabrielsen, pp. 207-235. New York: Harper & Row 1964 Stites, D.P., Caldwell, J., Carr, M.C., Fudenberg, H.H.: Ontogeny of immunity in humans. Clin. Imm. &Immunopath. 4, 519 527 (1975) Wara, D.W., Golbus, M.S., Ammann, A.J.: Fetal thymus glands obtained from prostaglandin-induced abortions. Transplantation 18, 387-390 (1974)

Accepted November 19, 1976

DNA-synthesizing cells in human fetal thymus.

Cell Tiss. Res. 178, 333-339 (1977) Cell and Tissue Research (~', by Springer-Verlag 1977 DNA-Synthesizing Cells in Human Fetal Thymus C. R6pke, B...
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