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Light and electron microscopy of the human fetal thymus.

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Light and Electron Microscopy of the Human
Fetal Thymus
JACK L. HAAR
Department of Anatomy, Medical College of Virginia, Health
Sciences Division, Virginia Commonwealth University,
Richmond, Viryinia 23298
ABSTRACT
The human fetal thymus was studied at stages from 9 to 20
weeks of gestation. At 9 weeks of gestation the human fetal thymus contained
lymphoid cells and was vascular although it was not yet lobulated nor did it
have a cortex and medulla. By 12 weeks the thymus was lobulated and at 14
weeks a cortex and medulla could be distinguished, although the medulla was
often more densely cellular than the cortex. By 18 weeks there were many lobules
and a mature looking cortex and medulla. Large lymphocytes at all stages of
thymus development studied were irregular in shape and often had blunt pseudopodia-like cytoplasmic extensions, or more slender cytoplasmic extensions. They
also often possessed numerous elongated mitochondria, a large Golgi complex
and strongly basophilic cytoplasm. Large lymphocytes were not attached to the
epithelial cells by desmosomes although some of the cytoplasmic extensions from
them were in association with extensions from epithelial cells. Primitive mediumsized lymphocytes at all stages studied were round in shape and had fewer mitochondria than the large lymphocytes. Epithelial cells were much less basophilic
than the lymphoid cells and usually contained aggregates of glycogen. Occasional
macrophages were observed within the developing thymus after 12 weeks of
gestation and one granulocyte was observed within the thymus at 9 weeks while
numerous granulocytes were seen within an interlobular septa at 14 weeks of
gestation. Vessels were present within the thymus at all stages studied and at
9 weeks some had a boundary between the blood and thymus which consisted of
only a thin endothelial cell and its basal lamina rather than the usual boundary
of an endothelial cell and its basal lamina plus an epithelial cell and its basal
lamina.
Although much effort has been directed
along similar lines using various research
animals (Ackerman, '67, Ackerman and
Hostetler, '70, Ackerman and Knouff, '64,
'65, Chapman and Allen, '71, Mandel, '70,
Weakley et al., '64), a detailed morphological study of the human fetal thymus
using fresh tissue taken early in gestation
has not been reported. Interest in the
thymus and its development was spurred
by the discovery of its importance in development of the cellular immune system
(Miller, '61, Metcalf and Moore, '71). It
was also suggested that the thymus of the
adult mouse was of importance in protecting malignant or auto-immune cells from
destruction by virtue of a blood-thymus
barrier (Clark, '63). Although much of the
ANAT. REC., 179: 463-476.
early work centered around the question of
intrathymic versus extrathymic origin of
lymphocytes, recent evidence has discounted an intrathymic origin in mice
(Moore and Owen, '67, Owen and Ritter,
'69). Furthermore, the yolk sac has been
strongly implicated as the organ from
which thymic lymphocytes arise in this
species (Tyan and Herzenberg, '68). Studies of hemopoiesis in the mouse yolk sac,
however have yielded little morphological
evidence to support such a theory (Haar
and Ackerman, '71).
Studies using human fetal thymus include a number in which immunoglobulin
production in the human fetus has been
undertaken (Hirokawa, '69, Hirokawa and
Received Nov. 8,'73. Accepted Feb. 14,'74.
463
464
JACK L. HAAR
Hatakeyama, '69, Pirofsky et al., '73, van
Furth et al., '65, '66). There have been,
however, but a few studies on the
ultrastructure of human fetal thymus
(Hirokawa, '69, Pinkel, '68).
The purpose of the present study was to
examine development of the human fetal
thymus earlier in gestation and from
fresher material than was used previously,
noting (1) development of the cortex and
medulla, ( 2 ) characteristics of early
thymic lymphoid cells, and ( 3 ) thymic
vasculogenesis.
MATERIALS AND METHODS
The fifteen fetuses which were used for
this research project ranged in gestational
age from 9 to 20 weeks as determined by
crown-rump lengths (Patten, '53) and
menstrual records. The number of specimens used included 2 for each of the following stages 9, 10 and 11 weeks; 3 at 12
to 13 weeks; 3 at 14 to 15 weeks; and
1 each at 16, 17 and 20 weeks of gestation.
The fetuses resulted from abortion and
sterilization by hysterectomy. Following removal of the fetus from the uterus the
thymus was extirpated, placed in fixative
at 4°C and, after 30 minutes, cut into
1mm3pieces and fixed for an additional 30
minutes. Karnovsky's fixative (25 ml of
8% paraformaldehyde, 5 ml of 50% glutaraldehyde diluted to 50 ml with 0.2 M
cacodylate buffer having a pH of 7.6) was
found to give best fixation. Following fixation, the tissue was postfixed for 2 hours
in similarly buffered cold 2% osmium
tetroxide; dehydrated and embedded in
durcupan (Fluka). One micron sections
were stained with 0.1% toluidine blue and
0.1% methylene blue in 1% sodium borate for 1 minute and studied with light
microscopy; thin sections stained with
uranyl acetate (Watson, '58) and lead
citrate (Reynolds, '63) were examined in
either an RCA EMU 3G or an Hitachi 12
electron microscope.
RESULTS
Cortex and Medulla
At 9 weeks of gestation the thymus is
enclosed by a continuous basal lamina
which separates it from the surrounding
loosely arranged bundles of collagen fibrils,
mesenchymal cells and occasional small
blood vessels (figs. 2-4). The organ is not
lobulated, nor is a cortex or medulla distinguishable at this early stage (fig. 1 ) .
Large lymphocytes and primitive mediumsized lymphocytes are present within the
thymus, however, and are easily discerned
with light microscopy as basophilic cells
(fig. 2). Most of the thymus cells adjacent
to the basal lamina are epithelial and
many appear columnar in shape, although
occasional lymphoid cells are seen in this
outer region (figs. 3,4). Dividing epithelial
cells and lymphoid cells frequently are
seen throughout the organ at this stage
(fig. 2 ) .
By 12 to 14 weeks of gestation the thymus has become lobulated with the developing connective tissue septa between
lobes frequently possessing blood vessels
(figs. 7, 8). Also at this stage, areas occupied by dense concentrations of basophilic
cells appear within the thymus; occasionally such areas are located in the cortex
but often they are in the medullary region
(fig. 7 ) .
At 18 weeks of gestation lobulation has
increased and the thymus has a morphology similar to that seen in the adult, viz.,
a cortex composed of a dense population
of basophilic cells, and a medulla commonly possessing Hassall's corpuscles and
having fewer lymphocytic cells than are
found in the cortex (figs. 9, 10).
Cells of the thymus
The epithelial cells of the thymus at the
stages studied are easily distinguished as
being less basophilic and their cytoplasm
less dense than lymphoid cells. At 9 weeks
of gestation some epithelial cells at the
outer surface of the thymus are columnar
in shape. These cells are joined to one another by desmosomes and contain aggregates of glycogen, numerous mitochondria
and rather short profiles of rough endoplasmic reticulum (figs. 2, 3, 4, 6). The
lymphoid cells can be divided into two
populations : large lymphocytes and primitive medium-sized lymphocytes. Large
lymphocytes which were observed at all
intervals from 9 to 20 weeks of gestation
are greater than 10 in diameter and are
irregular in shape. In addition, large lymphocytes display various types of cyto-
MORPHOLOGY OF HUMAN FETAL THYMUS
plasmic extensions, viz. blunt pseudopodialike extensions (figs. 6, 12), elongated
slender extensions (fig. 5), and short
slender extensions in association with
similar extensions from epithelial cells
(fig. 11). Large lymphocytes are quite
basophilic, usually have numerous elongated mitochondria, a large Golgi complex,
only occasional short profiles of rough
endoplasmic reticulum and a nucleus with
a fairly dense band of chromatin around
its perimeter plus one and often two nucleoli. Large lymphocytes most often are
near the outer surface of the thymus, and
separated from the basal lamina by only
a thin projection of cytoplasm from an
epithelial cell (figs. 4 , 6 ) . In one case there
exists a small gap between epithelial cells
at the location of a large lymphocyte with
long slender cytoplasmic extensions (fig.
5). Primitive medium-sized lymphocytes
within the developing thymus are round or
somewhat irregular in shape lacking the
cytoplasmic projections of the large lymphocytes, have dense basophilia, a slightly
increased number of polysomes when compared to large lymphocytes and lack a
prominent Golgi complex. In addition,
primitive medium-sized lymphocytes have
fewer and less elongated mitochondria
than do large lymphocytes and the nuclei
have a thinner band of peripheral chromatin and more often one rather than two
nucleoli (fig. 3 ) . Other cells which were
seen within the developing thymus included occasional macrophages seen after
12 weeks of gestation. A granulocyte was
first observed intrathyrnically at 9 weeks
of gestation and at 14 weeks granulocytes
containing large basophilic granules were
present in large numbers in an interlobular septa (fig. 8). These granulocytes have
not been previously reported in the human
fetal thymus. Additional data are required
on these cells before a full report can be
made on them.
Vessels
At 9 weeks of gestation there are extrathymic blood vessels associated with the
connective tissue fibers and mesenchymal
cells surrounding the thymus (fig. 2).
There are also intrathymic vessels at 9
weeks of gestation which are composed of
endothelial cells and a basal lamina plus
465
epithelial cells and their extraneous coat,
Numerous examples were found, however,
at this early stage of a structural boundary
between the blood and thymus which was
composed solely of endothelium and associated basal lamina (fig. 14). By 12-14
weeks of gestation, vessels are found
within the lobules of the thymus (fig. lo).
Some of these intralobular vessels are
characterized by endothelial cells which
possess numerous small vesicles lying adjacent to the lumen (fig. 13).
CONCLUSIONS AND DISCUSSION
Cortex and medulla
Studying the morphology of the earIy
human fetal thymus revealed both similarities and dissimilarities to development
of the thymus in research animals, A thymus early enough in gestation to be nonlymphoid is not included in this study,
although there is one at a stage prior to
differentiation of a cortex and medulla.
The examination of this early tissue suggests that the human thymus is vascular
even before connective tissue septa are distinguishable between lobes and before a
cortex and medulla differentiate. A cortex
and medulla are at first distinguishable by
observing the different densities of lymphocyte population. Unexpectedly, at 14
weeks when the medulla and cortex can
first be distinguished, the medulla has a
greater number of lymphoid cells than the
cortex indicating that cell division is a
characteristic of these medullary cells, This
finding suggests that the medulla when
first formed, may be the area of greatest
lymphocyte production and not the cortex
as in the adult.
Thymus cells
The earliest thymus studied was reremoved at nine weeks of gestation and
had no cortex or medulla although lymphocytes were present at this stage within the
mesenchyme surrounding and the parenchyme of the thymus. Of particular interest were the large lymphocytes with
pseudopodia. These cells resemble very
closely cells reported in the developing
chick thymus which have been described
as being motile (Leene et al., '73). These
observations suggest that the large lympho-
466
JACK L. HAAR
cytes may move between the organ and the
surrounding mesenchyme. No large lymphocytes were found passing through the
basement membrane surrounding the
organ as reported in the rabbit (Ackerman
and Hostetler, '70); however, the fact that
pseudopodia-containing large lymphocytes
are in close proximity to the basement
membrane suggests that this is the route
of passage. One large lymphocyte having
very long and slender cytoplasmic projections was found adjacent to a gap between
epithelial cells and thus very near the
basal lamina. This is the strongest evidence in this paper suggesting that in the
human being large lymphocytes pass
through the basal lamina. Perhaps the
slender cytoplasmic projections are necessary for passage through the basal lamina
and subsequently develop into the blunt
pseudopodia-like projections. Lymphoid
cells having pseudopodia suggestive of
their motility have not been previously reported in the human thymus. The close
cytoplasmic association between projections of epithelial cells and large lymphocytes suggest that the large lymphocytes
are held in place even though desmosomal
junctions do not exist. Granulocytes have
been reported as uncommon in the hamster, rare in the cat and rabbit, and common in the chick (Ackerman and Hostetler,
'70). Granulocytes in the human thymus
have been observed at 9 weeks of gestation and were a striking feature at 14
weeks although they have not been previously reported in the human thymus. Further information is needed before the significance of this finding can be interpreted.
are not composed of a typical four layered
structure. The concept of a structural nonpermeable blood-thymus barrier has, however, given way to a functional bloodthymus barrier to macromolecules (Raviola
and Karnovsky, ' 7 2 ) , thus reducing the
significance of the layers comprising the
boundary.
Vesicles within the cytoplasm of endothelial cells of fetuses at 14-18 weeks of
gestation corroborate the similar finding
reported by Pinkel ('68) in the human
fetus, and may be associated with transendothelial exchange. Large numbers of
vesicles have not been reported in monkey,
rabbit, mouse or chick thymic endothelial
cells.
Vessels
Vessels of the human fetal thymus have
previously been reported to be composed
of 4 layers: endothelial cells with a basement membrane and epithelial cells and a
basement membrane (Pinkel, '68). This
finding was consistent with that reported
by Clark ('63) in the adult mouse thymus
and postulated as a blood-thymus barrier.
In the fetal monkey, vessels of the thymus
occasionally were found which were incompletely surrounded by epithelial cells
(Chapman, '71). Unlike the vessels of the
older human fetal thymus, the vessels of
the younger thymus viz. 9 weeks gestation,
-
ACKNOWLEDGMENTS
The co-operation of the Department of
Obstetrics and Gynecology (Dr. Leo J.
Dunn, Chairman) at the Medical College
of Virginia is gratefully acknowledged. Appreciation is also expressed to Dr. William
P. Jollie for helpful criticism, Ms. Shirley
S. Craig for technical assistance and to
Ms. Kathy I. Forstner and Ms. Marilyn P.
Harrell for aid in manuscript preparation.
LITERATURE CITED
Ackerman, G. A. 1967 Developmental relationship between the appearance of lymphocytes
and lymphopoietic activity in the thymus and
lymph nodes of the fetal cat. Anat. Rec., 158:
387-400.
Ackerman, G. A., and J. R. Hostetler 1970
Morphological studies of the embryonic rabbit
thymus: The in situ epithelial versus the extrathymic derivation of the initial population
of lymphocytes in the embryonic thymus. Anat.
Rec., 166: 27-46.
Ackerman, G. A., and R. A. Knouff 1964 Lymphocyte formation in the thymus of the embryonic chick. Anat. Rec., 149: 191-216.
1965 The epithelial origin of the lymphocytes in the thymus of the embryonic hamster. Anat. Rec., 152: 35-54.
Chapman, W. L.,Jr., and J. R. Allen 1971 The
fine structure of the fetal and neonatal monkey (Macaca mulatta). Z. Zellforsch. Mikrosk.
Anat., 114: 220-233.
Clark, S. L.,Jr. 1963 The thymus in mice of
strain 129/J, studied with the electron microscope. Am. J. Anat., 112: 1-33.
van Furth, R., H. R. E. Schuit and W. Hijmans
1965 The immunological development of the
human fetus. J. Exp. Med., 122: 1173-1187.
1966 The formation of immunoglobulins by human tissues in vitro. 111. Spleen,
lymph nodes, bone marrow and thymus. Immunology, 11: 19-27.
Haar, J. L., and G. A. Ackerman 1971 A phase
MORPHOLOGY OF HUMAN FETAL THYMUS
and electron microscopic study of vasculogenesis and erythropoiesis in the yolk sac of
the mouse. Anat. Rec., 170: 199-224.
Hirokawa, K. 1969 Electron microscopic observation of the human thymus of the fetus
and the newborn. Acta. Pathol. Jap., 19: 1-13.
Hirokawa, K., and S. Hatakeyama 1969 Immuno-cytochemical study on the development
and differentiation of the immune system of
the human fetus. Acta. Pathol. Jap., 19: 151160.
Leene, W., M. J. M. Duyzings and C. van Steeg
1973 Lymphoid stem cell identification in the
developing thymus and bursa of Fabricius of
the chick. Z. Zellforsch. Mikrosk. Anat., 136:
521-533.
Mandel, T. 1970 Differentiation of epithelial
cells in the mouse thymus. Z. Zellforsch.
Mikrosk. Anat., 106: 498-515.
Metcalf, D., and M. A. S. Moore 1971 Embryonic aspects of haemopoiesis. In: Haemopoietic cells, p. 224-271 (Frontiers of Biol.,
Vol. 24).
Miller, J. F. A. P. 1961 Immunological function of the thymus. Lancet, 2: 748-749.
Moore, M. A. S., and J. J. T. Owen 1967 Experimental studies on the development of the
thymus. J. Exp. Med., 226: 715-725.
467
Owen, J. J. T., and M. A. Ritter 1969 Tissue
interaction in the development of thymus lymphocytes. J. Exp. Med., 229: 431442.
Patten, B. M. 1953 In: Human embryology.
McGraw-Hill Book Company, Inc. New York.
Chap. VII, 181-203.
Pinkel, D. 1968 Ultrastructure of human fetal
thymus. Am. J. Dis. Child., 115: 222-238.
Priofsky, B., G. H. Davies, J. C. Ramirez-Mateos
and B. W. Newton 1973 Cellular immune
competence in the human fetus. Cell. Immunol., 6: 324-328.
Raviola, E., and M. J. Karnovsky 1972 Evidence for a blood-thymus barrier using electron-opaque tracers. J. Exp. Med., 236: 466-498.
Reynolds, E. S. 1963 The use of lead citrate
at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol., 17: 208-212.
Tyan, M. L., and L. A. Herzenberg 1968 Immunoglobulin production by embryonic tissues;
thymus independent. Proc. SOC. Exp. Biol Med.,
128: 952-954.
Watson, M. L. 1958 Staining of tissue sections
for electron microscopy with heavy metals.
J. Biophys. Biochem. Cytol., 4 : 475-479.
Weakley, B. S., D. I. Patt and D. Shepro 1964
Ultrastructure of the fetal thymus in the golden
hamster. J. Morphol., 115: 319-354.
Abbreviations
C, large lymphocyte
E, epithelial cells
G, granulocyte
L, primitive medium-sized lymphocyte
M, mesenchymal cell
N. endothelial cell
R,’ erythrocyte
a, mitosis
b, basal lamina
c, capillary
f, collagen fibril
g, Golgi complex
m, mitochondria
p, cytoplasmic projection
r, rough endoplasmic reticulum
PLATE 1
EXPLANATION O F FIGURES
1
Section through thymus taken from a fetus a t 9 weeks of gestation.
This stage is characterized by having neither lobules nor a cortex and
medulla. A connective tissue capsule is forming around the periphery
of the organ and lymphoid cells are present within the organ appearing as very basophilic cells. X 268.
2 Higher magnification of the outer surface of the organ shown in
figure 1. Note the endothelial cells ( N ) of a capillary containing
erythrocytes ( R ) within the developing capsule. Within the thymus
there are light staining epithelial cells ( E ) , moderately basophilic
large lymphocytes ( C ) , strongly basophilic primitive medium-sized
lymphocytes ( L ) and one lymphoid cell in mitosis ( a ) . x 1000.
3
468
Electron micrograph of the same tissue shown in figures 1 and 2.
Note the continuous basal lamina ( b ) along the outer surface of the
thymus separating it from the developing connective tissue capsule.
Epithelial cells ( E ) containing glycogen aggregates surround a cluster
of three primitive medium-sized lymphocytes ( L ) . These lymphocytes
are round or slightly irregular in shape, have occasional profiles of
rough endoplasmic reticulum ( r ) , numerous polysomal clusters and
their mitochondria ( m ) are primarily round. X 8,900.
MORPHOLOGY OF HUMAN FETAL THYMUS
Jack L. Haar
PLATE 1
469
PLATE 2
EXPLANATION OF FIGURES
4 70
4
Electron micrograph showing developing capsule region and outer
surface of thymus at 9 weeks of gestation. A portion of a large lymphocyte (C’) and a granulocyte ( G ) with a few small granules are
seen among projections from mesenchymal cells (M). A basal lamina
( b ) may be observed and a large lymphocyte ( C ’ ) is evident within
a developing thymic capillary. This cell and capillary may be seen
to better advantage in figure 12 taken from a n adjacent section. The
outer thymic epithelial cells( E ) have a columnar shape and contain
glycogen. x 5,880.
5
A portion of a large lymphocyte ( C ) having long slender cytoplasmic
extensions ( p ) and located just within the thymic basal lamina ( b ) .
At one point (arrow) a gap exists between two epithelial cells ( E )
so that only the basal lamina separates the large lymphocyte from
the developing capsule. x 16,200.
6
This large lymphocyte ( C ) is separated from the developing thymic
capsule and basal lamina ( b ) by only thin cytoplasmic projections of
the epithelial cells ( E ) . The large lymphocyte has blunt pseudopodialike cytoplasmic projections ( p ) , two nucleoli within the nucleus,
basophilic cytoplasm, elongated mitochondria ( m ) , a well developed
Golgi complex ( 9 ) and only occasional short profiles of rough endoplasmic reticulum ( r ) . X 5,175.
MORPHOLOGY OF HUMAN FETAL THYMUS
Jack L. Haar
PLATE 2
471
PLATE 3
EXPLANATION OF FIGURES
7
Light micrograph of a thymic lobule a t 14 weeks of gestation. Mesenchymal cells (arrows ) within the developing capsule, intralobular
capillaries ( c ) and interlobular capillaries are evident. At this stage
the cortical region of the lobule is less densely cellular than is the
medulla. x 190.
8
Higher magnification of a n interlobular area adjacent to the lobule
shown in figure 7. Present within the interlobular area are several
granulocytes (arrows ). A capillary containing several erythrocytes
( R ) is present within the mesenchyme surrounding the thymus. Epithelial cells ( E ) and developing lymphocytes (L) can be seen within
the thymus. x 1000.
9
Light micrograph of thymus at 20 weeks of gestation showing lobulation and a cortex which is much more densely cellular than the
medulla. x 140.
10 Higher magnification of a portion of a lobule shown in figure 9. A
capsule forming around the lobules is easily seen as are developing
capillaries ( c ) within the thymus. X 400.
11 Electron micrograph of a large lymphocyte within a thymus at 20
weeks of gestation. This cell is separated from the outer basal lamina
( b ) by only thin cytoplasmic projections from epithelial cells (E).
Processes from mesenchymal cells (arrows) and collagen fibrils ( f )
are seen surrounding the thymus. Cytoplasmic projections ( p ) from
the large lymphocyte appear to be in association with the epithelial
cells, Mitochondria ( m ) , a few strands of rough endoplasmic reticulum ( r ) basophilic cytoplasm and a prominent Golgi complex ( 9 )
are features of this cell. X 19,400.
4 72
MORPHOLOGY OF HUMAN FETAL THYMUS
Jack L. Haar
PLATE 3
PLATE 4
EXPLANATION OF FIGURES
12 A section adjacent to the one in figure 4 at 9 weeks of gestation.
The granulocyte ( G ) with abundant rough endoplasmic reticulum
slender cytoplasmic projections (arrows) is seen in association with
projections from mesenchymal cells ( M ) and collagen fibrils ( f )
outside the basal lamina ( b ) enclosing the thymus. The large lymphocyte ( C ) with blunt cytoplasmic projections ( p ) is within a
developing intralobular capillary, the portions of endothelial cells ( N )
shown being more basophilic than the epithelial cells ( E ) . x 7,100.
13 A blood vessel from within a thymus at 18 weeks of gestation containing a nucleated erythrocyte ( R ) . Note the numerous vesicles
(arrows) within the cytoplasm of the endothelial cells and the cytoplasmic projections ( p ) from these cells into the lumen of the vessel.
X 14,500.
14
474
This electron micrograph shows a blood-thymus boundary at 9 weeks
of gestation consisting of only endothelial cells ( N ) and a basal
lamina ( b ) . This boundary separtes erythrocytes ( R ) within the
vessel from a large lymphocyte ( C ) outside the vessel. A portion of
an epithelial cell ( E ) is also present. X 13,800.
MORPHOLOGY OF HUMAN FETAL THYMUS
Jack L. Haar
PLATE 4
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