Paper - Hematopoiesis in young human embryos
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Hematopoiesis In Young Human Embryos
Willlam Bloom And G. W. Bartelmez
Department of Anatomy, The University of Chicago, Illinois
Four Plates (Eleven figures)
- This work has been aided by a grant from the Dr. Wallace 0. and Clara. A. Abbott Memorial Fund of The University of Chicago. We are indebted to Drs. F. Adair, J. I. Brewer, H. Jones and A. Kantor for the opportunity of studying certain of these young embryos.
The observations reported here show that although blood cell formation in its early stages in the human embryo and especially the yolk sac follows the same general lines as in other mammals, it does not proceed in exactly the same fashion as in any other species which has been thoroughly studied. Practically all of the young human ova previously described are inadequately prepared from the hematological point of view, as they were usually fixed in formalin and in most cases a considerable lapse of time occurred after removal of the tissue before it was fixed. The staining has usually been hematoxylin and cosin, which does not permit a sharp discrimination of the various types of blood forming cells.
The present study is based on a number of excellently fixed and stained young human ova with their yolk sacs. In all cases, the embryos are normal and were fixed very promptly in formalin-Zenker; in all of them. the chromosomes are sharply outlined. These ova have been prepared so well that the material is as favorable as that obtained from the usual laboratory animals. After studying this material we believe that the essential parts of the morphological picture of hematopoiesis in the human yolk sac are now apparent.
We believe this conclusion will hold regardless of the interpretation that may be placed by other investigators on the potentialities of ‘the cells involved. As will appear in the following. pages, the first evidences of hematopoiesis are to be found in early primitive streak stages. With the appearance of "the head process, hematopoiesis is definitely established.
The early stages described here‘ are from material of The University of Chicago -Human Embryological Collection. The later stages are from the collection of the late Professor Maximow; they were mentioned brieﬂy by him (’27) and in more detail by Bloom (’38 b-), in their reviews of mammalian embryonic hematopoiesis. The serial sections made after embedding the ova in paraffin, paralfin—celloidin', or celloidin were stained with hematoxylin and eosin or hematoxylineosin-azure II. Except in the case of ' the two youngest specimens, all or part of each yolk sac was stained with hernatoxylin-eosin-azure II.
To avoid-repetition during the description of the sections the cell types found in this material will be described -here, (and some of the more important synonyms will be given). References will be made to particular cells as seen in the accompanying figures, as these drawings, very accurately made by Miss E. Bohlman, convey more graphically than words the appearance of the cells.
The cells which we label as hemocytoblasts occur intra- and extravascularly (cell 1 in all figures). In the sections they appear as spherical or slightly polygonal cells often provided with ameba-like processes. The cytoplasm is deeply basophil and may contain a few small vacuoles. An acidophil attraction sphere is occasionally visible. The relatively very large nuclei are spherical or somewhat indented. The very large and acidophil nucleoli may be single or multiple and‘ are often very angular. ‘The finely granular chromatin is aggregated in larger masses along the nuclear membrane in some cells. These cells are identical in appearance not only with the primitive blood ‘cells (see next paragraph) but also with the large free basophil stem cells seen in all hematopoietie foci of both embryonic and adult man; that is, both myeloblasts and large lymphocytes.‘ Some few of the hemocytoblasts are the size of medium sized lymphocytes of lymphatic tissue (cells 19, 20) and occasionally even very small ones occur (cell 23). Such cells in bone marrow are called micromyeloblasts by some hematologists. It is on the question of the i.denti.ty of these basophil cells in various hematopoietie situations that so much of the hematological controversy has centered in the past (see detailed discussion of the hematopoietic stem cells in Bloom, ’38 a, p. 416). As pointed out there, none of the morphological criteria for the discrimination of the various stem cells holds in all cases, and the identification of these stem cells is usually made on the cells they give rise to. This is not a demonstration of morphological differences. For this paper we shall merely restate our conclusion that these cells are not only morphologically but also potentially identical in all of the situations in which they occur. We believe this conclusion also follows for the human yolk sac, from the data presented here. The recent claim of Rich and co-workers ('39) that.the stem cells can be discriminated by their types of motility in tissue culture awaits confirmation.
Primitive blood cells
These are the first free precursors of blood cells and are morphologically identical with hemocytoblasts. Because of this morphological identity Ferrata (’18) called them transitory hemocytoblasts. Before the papers of Bryce (’04), Dantschakoff (’08), Maximow (’09), J olly (’23), it was generally believed that all of the primitive blood cells would become erythroblasts. But this is incorrect, as these authors have shown and as can be seen from the data presented here.
Lymphoid wandering cells
This name was given by Maximov ("09) to cells of lymphocyte, that "is hemoeytoblast, type which he found moving in the mesenehyme. They correspond morphologically to various sized lymphocytes (that is, hemocytoblasts) and have small amounts of ameboid cytoplasm (staining faintly blue with azure in the smaller cells and deeply blue in the larger ones) and nuclei with large nucleoli. If the group is limited to the small cells, this is an unimportant one here.
This is the largest group of cells in the blood cell forming-areas. The youngest ones closely resemble and are connected by many transition forms with the hemocytoblasts. The nuclei become smaller, the nucleoli smaller and often more numerous, and the chromatin particles more prominent as the cells mature. The cytoplasm remains about the same size but loses its basophilia as: hemoglobin accumulates in it. During the process the cytoplasm varies in color from blue to pale gray to brownish gray to gray-orange to a bright orange in the final stages. These have been called basophil (cells la), polychromati.c (cells 2, 3), and orthochromatic primitive erythroblasts (cells 4). The final stage, which in man is usually a nucleated cell, might also be called a primitive erythrocyte. (cells '4), especially in figures B and G. All of the stages of polychromatophil and orthochromatic primitive erythroblasts often contain one or more refractile brownish bodies (cell to left of G). It is, for us,“ practically impossible to separate a distinct group of basophil erytliroblasts in the primitive generation of red blood cells of man. Some of the hemocytoblasts become especially basophil and develop more prominent and heavier chromatin granules (as cells la) but there are so many transitions between such cells and the heinocytoblasts on one hand and the early polychromatic erythroblasts on the other that they hardly form a morphologically distinct cell group.
The definitive erythroblast series also begins with cells of hemocytoblast type. Occasionally they become especially basophil (definitive basophil erythroblasts). Then the cytoplasm becomes paler and the chromatin assumes a more regular distribution with the nucleoli smaller and more numerous; this is often spoken of as the checker board nucleus‘ (cells 7 and 8, fig. B). As the cells mature the cytoplasm goes through the same staining changes as in the generation of primitive erythrocytes until it is finally orthochromatic (cell 9, fig. B). The nucleus becomes progressively smaller and pyknotic and is finally extruded to produce the mature definitive erythrocyte. Mitoses are found in all of the definitive erythroblasts (as in cell 7’).
Some of the cells of this group, especially the larger ones, have all of the morphological features of macrophages (histiocytes, clasmatocytes, resting wandering cells, etc.) of the adult organism. When rounded-——as they are within the yolk sac vessels—-the eccentric, rather pale staining nucleus is more or less indented, the nuclear membrane usually irregular, and the nucleoli small; the foamy or finely reticular cytoplasm may (cells 11) or may not contain ingested material. These first intravascular phagocytes were called endothelial phagocytes by Maximow (’09), who traced them to the primitive endothelium in the yolk sac. Some of them also occur in the mesenchyme, usually without included material (cell 10, fig. B). These latter are the histioid Wandering cells of Maximow. The name macrophage seems better adapted to both types (although it does ' not connote po‘h=.ntia.li‘r.ies other. than toward phagocytosis).
In addition to these large characteristic macrophages there are smaller cells of a variety of appearances which should be placed in this category. These cells vary from but slightly altered, typical large and small lymphocyte-like cells (the cells called hemocytoblats here) to cells which show all the stages of transformation up to typical macrophages. Some of these cells are shown in the accompanying figures. Cell 16 is a typical hemocytoblast which has become phagocytic, cells 18 and 21 have the nuclei of medium-sized lymphocyte (i.c., hemocytoblasts) but have more cytoplasm than‘ is usual with these cells. Of these two cells, 18 contains phagocytosed material, while 21 does not. These but slightly altered lymphocytes gradually merge into the macrophages. Cell 24 has been figured to show a stage about half-way between lymphocyte (hemocytoblast) and macrophage. These small phagocytes and the cells from which they originate are identical with the macrophages arising from the blood agranulocytes as they occur in early stages of inﬂammation in the body and also in cultures of blood, lymph, and lymphatic tissue. (see review by Bloom, ’38 c).
These vary from typical but enlarged primitive erythroblasts (cell 22) and erythrocytes to cells twice the size of primitive erythrocytes. They may have one or several distinct, smoothly outlined, oval or round nuclei with small nucleoli" and an even, acidophil cytoplasm. These cells are not to be confused with either macrophages or megakaryocytes. Nor are they like any of the normal cells of the various types of connective tissue. Cell 15 is an example of the largest of these atypical giant cells; it is perhaps of primitive erythroblast origin.
These are very infrequent in the human yolk sac although numerous in those of some other mammals, especially the cat. In this human material there are a few atypical large cells with nuclei resembling those of young developing megakaryocytes (cell 12, plate 2). Specifically fixed and stained preparations as those of Downey or Kingsley are not available in this rare human material.
These infrequent cells_in this material do not merit an extended description. The granules of the eosinophil (cell 13) and heterophil (cell 14) types are quite characteristic, although in the latter they are almost at the limit of visibility. The bas.ophil leucocytes require absolute alcohol fixation. Some of the myelocytes are much like those of the bone marrow with their large nucleoli. A few of them look as though they came from mesenchymal cells Without passing through a hemocytoblast stage, for they lack large nucleoli. Four such cells were figured by Bloom (’38 1); plate IV, 13- 384) and will not. be reproduced here.
Primitive Streak Formation
Beginning with primitive streak formation. (H1496; see Brewer ’37; ’33)- This is the earliest Well-preserved ovum in our series; it is about the same age as the Peters ovum, that is, probably 13 days. The trophoblastic mesenchyme consists of out-stretched spindle or star-shaped mesenchymal cells with here and there rounded, large macrophages. The latter correspond with the histioid wandering cells of Maximow (’09,’27). There is no evidence of hematopoiesis; cells of hemocytoblast appearance are absent. Nor is there anything which by any criterion could be called “angioblast” in a hematopoietic or a vasoformative sense. There are no blood vessels.
The yolk sac at this time is a vesicle lined by a continuous single layer of predominantly squamous epithelial cells among which, particularly where they adjoin the primitive ectoderm, are irregularly spheroidal cells. The entoderm is invested with a - thin, irregular, often net-like sheet of stellate and squamous cells (fig.'H, extra-embryonic mesoderm) which are occasionally in continuity with the magma strands of the chorionic cavity. At the caudal end of the embryo these yolk sac mesodermal cells, which will give rise to the yolk sac mesenchyme and mesothelium, are continuous with the rest of the extraembryonic mesoderm; that is, of the belly stalk, amnion, chorion and magma. The cells of the yolk sac _mesoderm close to the embryonic disc form a loose net work which ﬂattens into a. fairly regular squamous. epithelium distally. These irregular, often spindle shaped mesenchymal cells have large oval nuclei with finely divided chromatin and pale staining small nucleoli. The outer cells of this network will become the yolk sac (splanchnic) mesothelium, while some of the innermot ones are in all probability the source of future hematopoiesis (and local angiogenesis). There are no blood vessels and no collections of free cells such as occur in later stages and become precursors of hematopoietic foci.
The yolk sac cavity contains a few degenerating cells of unidentifiable nature. There are numerous wandering cells of macrophage type outside the yolk sac.
There is a small collection of large and small macrophages in the amnionic mesenchyme close to its attachment to the embryonic disc.
It is unfortunate that well prepared specimens from the next 3 or 4 days are not available, for during this period the mesenchyme develops and begins to give rise to blood cells in the yolk sac.
Early head process
(H 1459); see Jones and Brewer, ’35; Brewer, ’40). This is our next older stage and is about 18% days old.. Hematopoiesis is just beginning in the yolk sac close to the embryonic disc (fig. I) and is well advanced at the distal pole of the sac (fig. J). The entodermal lining of the yolk sac is a. fairly regular epithelium and the lumen eontains a few macrophages. The mesoderm covering the amnion and yolk sac is very irregular and in places extends for some distance into the chorionic cavity. For the most part, however", the yolk sac covering (splanchnic mesothelium) is more epithelium-like than mesenchymatous. Between the entoderm and mesothelium are numerous outstretched mesenchymal cells and blood sinuses. Some of the latter are empty while others contain rapidly proliferating hemoeytoblasts and primitive erythroblasts. It is often difficult to separate the cell types in these prepa.rations stained with hematoxylin and eosin, but those with deeply indented nuclei and relatively large nucleoli are hemocytoblasts. There are a few extravascular hemocytoblasts. In a few vessels the hemocytoblasts are closely paeked and seem to be in contact. This is probably an indication of rapid proliferation (fig. I). The same phenomenon is also observed at slightly later stages (fig A, 1) and is of common occurrence in the yolk sacs of mammals and birds. In a few areas of extensive blood cell formation we cannot tell whether distinct vessel walls are present and whether hematopoiesis here occurs within vessels or merely in spaces in the mesenchyme (see fig._ J). Other cell. types seem to be absent as far as can be seen in these slides. There are empty blood vessels in the chorionic mesoderm and in the villi; three or four lymphoid wandering cells are seen in the chorion plate, but no hematopoiesis. In one section there are two small collections of syneytium-like mesoblasts in the belly stalk. There is also one vessel segment, extending through three sections, with primitive erythroblasts beginning to degenerate. There is one typical extravascular hemocytoblast. These are the only areas of hematopoiesis in the blastecyst exclusive of the yolk sac. There is no evidence in this embryo of the invasion of the yolk sac and belly stalk by a vasoformative tissue. Villi in all parts of the chorion contain isolated vascular primordia. VVe find no evidence for the origin of vascular primordia from the cytotrophoblast. All of our observations for these early stages point to multiple foci of development of vessels through direct transformation of mesenchymal.cells in the chorion, belly stalk and yolk sac.
(H1515, comparable to Heuser, ’32). This ovum is very well preserved and is 19 days old. Part of the yolk sac is stained with hematoxylin-eosin—azure II. The rest of the ovum is stained with hematoxylin and eosin.
The yolk sac is now ballooning out into a thin-walled vesicle of mainly two epithelial layers between which are small foci of hematopoiesis.
The chorion and villi do not show quite as much vascularization as the previous specimen (H1459). The primitive vessels are empty. Two hemocytoblasts are seen in the mesenehyme of the villi. There are a few atypical giant cells in the belly stalk; one of them suggests an early megakaryocyte with its folded nucleus. A large isolated segment of a vessel in the belly stalk contains several macrophages of various sizes, two hemocytoblasts, but no erythroblasts.
The entoderm of the yolk sac in this specimen is much higher than in the younger ova and now forms an irregular, low columnar epithelium. The cytoplasm is unevenly vacuolated and the nuclei contain one or more small but prominent nucleoli. A brush border is not apparent. The yolk sac mesothelium is decidedly lower than the entodermal epithelium although it too consists of markedly vacuolated cells. In the distal part of the yolk sac wall hematopoiesis is well advanced; it is much less prominent close to the embryonic body. The ab-embryonic portion of the yolk sac is adherent to and fused "with the trophoblastic mesoderm for a short distance.
As the wall of the yolk sac approaches the embryonic body it narrows down to a thin strip of tissue consisting mainly of two layers of epithelium with here and there a few spindle-shaped (mesenchymal) cells between them. There are also a few scattered foci of deeply basophil cells with large nucleoli (fig. D). These are hemocytoblasts; they are the primitive. blood cells of most authors and are the immediate source of blood cell formation. Morphologically they are hemocytoblasts (large lymphocytes) with their relatively large nuclei, large nucleoli and densely basophil cytoplasm (fig. D), and We shall call them so for the reasons given above under “Nomenclature.” They are apparently not within blood vessels, although they are more or less. completely surrounded by mesenchymal cells. This part of the yolk sac also contains small empty blood vessels, apparently as isolated spaces.
These free basophil cells (hemocytoblasts) multiply by mitosis and form larger groups; then some of them begin to develop into primitive erythroblasts. The next stages in this process can be followed in the more distal parts of this yolk sac, where the hematopoietic process is -more advanced and more extensive.
In this part of the yolk sac hematopoiesis is predominantly intravascular and is represented by a variety of cell forms. Mot of them are various stages of polychromatophil erythroblasts of the primitive generation. Some few of them are practically mature primitive erythrocytes with orange staining cytoplasm and a pyknotic nucleus. Much less numerous are cells of hemocytoblast type. Most of these.are relatively large cells of approximately the size of the primitive erythroblasts but differing from them by their deeply basophil cytoplasm and very large multiple or single nucleoli. A few of the lymphoid cells (hemocytoblasts) are about the size of the medium-sized lymphocyte (cells 20, plate 2) and there. is even an occasional one which is identical in appearance with the small "lymphocyte of lymphatic tissue (cell 23). Although some of the hemocytoblasts occur extravascularly, most of them are Within vessels.
There are numerous transition forms between the hemocytoblasts and the primitive erythroblasts, so that in the case of many early cells in this series it is impossible to allocate them to one group or the other. A similar difficulty is met in the case of transition forms between the hemocytoblasts and the small phagocytes in these vessels.
Less ‘numerous than the hemoeytoblasts are the small phagocytes. These are very polymorphous and are suggestive of the early changes in the hematoge-nous non-granular leucocytes in inﬂammation. That is, the nuclei in some are typical lymphocytic nuclei but occur in cells with slightly increased amounts of cytoplasm. In other cells, usually with more cytoplasm which is often vacuolated, the ch.romatin is decreased a.nd the nucleoli are decidedly smaller. Some of these cells have deeply indented nuclei. These are early phagocytes and not infrequently containingested material (cell 18). There are even occasional phagocytes which would be typical hemocytoblasts with deeply basophil cytoplasm but for their phagoeytosed material (cell 16).
Many of the hemocytoblasts, even Within blood vessels, show numerous pseudopodia; these probably are the effect of the fixation agent. The primitive erytl1robla.st_s and- erythrocytes "do not‘ have such processes.
The vessels in this yolk sac contain a few -atypical cells (cell 22) whose nature is undetermined. In some respects they suggest giant primitive, polychromatopliil erythroblasts.
Mitoses are common among the hemocytoblasts and the primitive erythroblasts. The endothelial cells resemble those of the adult vessels and are identical with the mesenchymal cells lying between the vessels and the yolk sac epithelium. The endothelial cells practically never- show phagocytcic activity in this embryo, although this is evident at slightly later stages. Extravascular lymphoid Wandering cells are exceedingly rare".
A point of some note is the appearance of degenerating primitive erythroblasts; these show a very granular cytoplasm and in a few cells karyolysis. This is perhaps more marked in later ova.
- It is hard to distinguish primitive basophil erythroblasts as a distinct cell group. _The transformation into polychromatophil erythroblasts is probably very rapid. There is an occasional endothelial cell which is bulging into the lumen and perhaps this is evidence of development of a hemocytoblast from primitive endothelium. But the cells suggesting this are notas convincing as several seen in H1516 (see below) or int he illustration by Maximow (’09) of the rabbit yolk sac endothelium.
- It is obvious from the study of this yolk sac that the first hemocytoblasts do not all develop into primitive erythrocytes, for, in addition to this transformation, some of the hemocytoblasts persist as such While others become phagocytes .or giant cells. As will be shown below in older embryos, still others become megakaryocytes and granulocytes.
(H1516; copulation age 25 days.) This is the most beautifully preserved early embryo in our "series. The entire ovum is stained with hematoxylin-eosin-azure II. The vessels of the villi contain polychromatophil primitive erythrobla.sts of various stages of maturity and an occasional hemoeytoblast. There is no hematopoiesis in the mesenchyme of the villi.
There are a few minute extravascular foci of hematopoiesis in the belly stalk. Of these, two consist of normal primitive polyehromatophil erythroblasts, while in the others the cells are degenerating. One extravascular eosinophil myelocytr is developing directly from a mcsenchymal cell.
The yolk sac entoderm consists of irregular low columnar epithelial cells with vacuolated cytoplasm, often with clumps of basophil material which may be glycogen. The large relatively pale staining nuclei have small but prominent nucleoli.
There are several places in the yolk sac in which the earliest stages of hematopoiesis are developing extravascularly (fig. 0). In these areas there are large accumulations of deeply basophil cells of hemocytoblast type (cell 1). They seem to be in ameboid movement and many are undergoing mitotic division (cell 1’). The extravascular location of these cells is verified by tracing them through serial sections.
In a few places it is difficult to determine whether these large, irregular masses of "cells are 'intra- or extravascular. It is probable that these hemocytoblasts are being brought into communication with the lumina-of developing vessels. This process has been described by Maximow, Sabin (’20) and others. Not all of the -details are present in this one yolk sac, but the conclusion is undoubtedly correct that the majority of these hemocytoblasts will come to lie Within blood vessels and become intensively hematopoietic. Outside the vessels are scattered spindle shaped mesenchymal cells and a few Wandering cells of large and small hemocytoblast type. There are also occasional wandering cells of macrophage type (histioid Wandering cells of Maximow). In the yolk sac wall close to the embryo are numerous clearly extravascular hemocytoblasts which are identical in appearance with the intravascular ones. There are occasional cells which are obvious transition forms between outstretched mesenchymal cells and hemocytoblasts. There are‘ a few extravascular primitive erythrocytes singly or in pairs.
The most striking feature of the yolk sac is the very extensive intrat-vascular hematopoiesis. It is characterized by the presence of large numbers of hemocytoblasts with their basophil cytoplasm a.nd one or several large angular nucleoli, and primitive erythroblasts. The‘ hemocytoblasts usually occur singly, but sometimes they are in dense masses in which cellular borders are present but not sharp (cell 1, fig. A). "A few cells represent transitions between the hemocytoblasts and the primitive erythroblasts and may perhaps be termed basophil erythroblasts in that they have a_ deeply basophil cytoplasm and a chromatin pattern of their nuclei which is heavier than that of the hemocytoblasts and more like that of the primitive erythroblasts (cell la, figs. A, C). These basophil cells merge gradually into the series of polychromatophil primitive erythroblasts (cells 2, 3) which end with the orthochromatic primitive erythroblasts (cell 4). All of the above cell types show frequent mitoses. The hemocytoblasts are also developing in small numbers intravaseularly into atypical giant cells. The vessels, in addition, contain a very few small phagocytes and an occasional myelocyte.
There are a few degenerating erythroblasts with pyknotic nuclei and vacuolated cytoplasm. These are usually of the later polychromatic stages and are perhaps less numerous than in the specimen in the head process stage. Some of the free phagocytes and several endothelial cells contain ingested primitive erythrocytes. Two endothelial cells are deeply basophil and rounded and are probably turning into free cells of hemocytoblast character. Mitoses are not infrequent in the endothelium.
At one pole of the yolk sac are some isolated vascular loops apparently "not connected with the circulating blood of the rest of the vascular system and filled with degenerating erythroblasts.
This is the first embryo in this series in which there is a circulation. The circulating cells of the embryo proper eonsist of polychromatophil primitive erythroblasts in all stages, a much smaller number of large and small hemocytoblasts, a few macrophages and a very rare granulocyte. The small hemocytoblasts are the size of medium and small lymphocytes of the adult. Orthochromatie primitive erythroblasts (erythrocytes) are rare. The cytoplasm of these primitive erythroblasts is usually more granular than that of similar cells in the yolk sac. This is possibly due to differences in the rate of penetration of the fixative or perhaps to the paraffin-celloidin embedding of the embryo. The circulating blood also contains an occasional giant cell and some large primitive erythrocytes with two. nuclei. One giant cell in the lumen of the right aorta suggests an atypical megakaryocyte.
The mesenchyme of the body consists of typical polymorphous mesenchymal cells. Scattered among them are cytoblasts and macrophages (histioid wandering cells) With nucleoli much larger than those of the mesenchymal cells. Some of these wandering cells contain phagocytosed cellular debris. There are many mitoses in the mesenehymal cells. Transitions between mesenchymal cells and lymphoid wandering cells are frequent. In the whole series there are a few definitely extravascular primitive erythrocytes and heterophil myelocytes.
Hematopoiesis in the Yolk Sac of Older Embryos
Embryo of sixth week; 10.2 mm. crown-rump length. (H1371). The yolk sac of this embryo is another favorable specimen for ‘our study. The mesothelium now has a very distinct brush border. The entodermal cells have much chromophil substance in irregular clumps, similar to that in the cells of figure B. Hematopoiesis consists primarily in. the further production of primitive erythroblasts and erythrocytes, with numerous mitoses in these cells. Some hemoeytoblasts are also present but are not as numerous as in H1516, although a few small vessels-contain three or four of them. Scattered through the series are a few mediumssized intravascular phagocytes with an occasional atypical megakaryocyte.
Outstretched and roughly spindle-shaped mesenchymal cells form a thick, irregular layer between the epithelia and the vessels situated between them.
The primitive erythrocytes in this yolk sac show an extreme variability in shape and to-some extent in size, in contrast to the relatively regularly spherical shape of these cells in the earlier specimens. None of the primitive erythrocytes is as small as the definitive orthochromatic erythroblast (normeblast). This yolk sac also contains" a few scattered, primitive erythrocytes definitely extravascular in the mesenchyme; two are in mitosis. Perhaps the most striking change in this yolk sac is the occurrence of hemocytoblasts within the entodermal epithelial cells. Most of these are rounded and are sometimes contained" in a distinct vacuole. These hemocytoblasts move from one cell to another; indeed, two of them are seen migrating side by side from one cell to another. These intra-entodermal cells are not as numerous as in some of the later embryos where they develop into definitive erythroblasts. They will be discussed below at greater length.
Beginning seventh week; 12/mm. orown--rump length (H147 5). It is an excellently prepa.red specimen. Most of the blood cells are primitive erythrocytes. Primitive erythroblasts are much less numerous than before. The picture is otherwise unchanged except: (1) Mitosis occurs in the intra-entodermal hemocytoblasts and (2) in one place there are a few definitive late polychromatic erythroblasts (definitive normoblasts).
Fifteen and two-tenths millimeter
(H1412). The nuclei are fairly well preserved .but the cytoplasm of most of the cells is granular. The primitive erythrocytes dominate the picture; there is an occasional mitosis in them. There are. some scattered circulating definitive normoblasts. ' Eosinophil and heterophil myelocytes occur sporadically intra- and extravascularly. There a.re a few circulating hemocytobla_sts. Small and large groups of definitive erythroblas.ts (normoblasts) occur between and in the entodermal epithelial cells. A similar process was described by Saxer (1896) in the entoderm of the yolk sacs of sheep, cat and pig embryos.
(Maximow H9.) At this stage and slightly later there are several well-prepared embryos. In these specimens the yolk sac entoderm is much taller and frequently contains large masses of basophil substance (perhaps glycogen) (fig. B). In the yolk ac of the 20—mm. specimen there is a great increase‘ in the amount of mesenchyme between the entodermal epithelium on the surface and the epithelial outpouchings (de.sc_ribed in some detail by Jordan, '10). This meenchyme consits primarily of typical mesenchymal cells among which are scattered macrophages (histioid wandering cells) (cell 5, fig. B), a very occasional eosin-ophil myelocyte and numerous foci of definitive erythroblasts in various stages of development and also of degeneration. These will be described below in. greater detail.
The vessels are filled for the most part with circulating blood consisting mainly of mature, nucleated primitive erythrocytes among which is a ra.re hemocytoblast and even more rarely a macrophage or cosinophil leucocyte or myelocyte.
Some of the vessels are the site of an intense production of definitive erythrocytes (cells '7, 8, 9, fig. B). This series of permanent oxygen carriers begins with large basophil cells of hemocytoblast type and extends through the whole series of definitive polychromatophil erythrobla.sts and orthochromatic erythroblasts (the normoblasts of many authors). This series is identical with that found in the later embryonic foci of hematopoiesis in the liver, bone. -marrow, and spleen and with that found in adult normal bone marrow. The stages in this transformation are shown in the accompanying figure B. The cells of the definitive series differ primarily from those of the primitive" series by their much smaller size and by the more obvious checkerboard appearance of their nuclei.. There is no evidence whatever of the development of the smaller, definitive erythrocytes from the‘ large primitive ones. Both lineages undergo a parallel series" of cytoplasmic changes indicative of progressive accumulation of hemoglobin, evidenced in thee preparations by progressive polychromatophilia and finally orthoc-hromatophilia. It is only in the definitive series that the large immature cells of hemocytoblast type become much smaller through progressive division and maturation; and the shrunken, pyknotic nuclei are eventually extruded. These diiferences appear in figures A and B.
In addition to the foci of definitive erythropoiesis within the vessels and in themesenchyme, there are large foci of developing definitive erythrocytes within the entodermal epithelial cells. It is possible to trace the development of these definitive _erythroblasts from" large basophil cells of hemocytoblast type. There is no evidence of these hemocytoblasts developing from the entoderm. A.meboid hemocytoblasts were twice seen entering the entoderm from the mesenchyme. "The definitive erythroblasts in the entoderm multiply frequently and die. These are abortive islands of definitive erythropoiesis a.nd the cells produced do not get into the circula.ting blood.
Hematopoiesis does not occur in the chorion or villi of this embryo.
“Nine weeks embryo.” (Maximow H29.) In the yolk sac of this embryo there is very little hematop-oiesis; The vessels are filled with primitive erythrocytes and a- few definitive normoblasts. They also contain numerous foci of macrophages with ingested erythrocytes and green and yellow pigment (cell 11, fig. Gr).
As the amniotic cavity increases rapidly .at the beginning of the third month of development and the extra-embryonic coelom is correspondingly reduced," the yolk sac is usually compressed between amnion and chorion and this presumably reduces its blood supply. This may be a more important factor in the regression of yolk sac hematopoiesis than an inhibition from the liver where active blood cell formation had begun in the 6-week stage.
As the embryos become older, hematopoiesis gradually subsides in the yolk sac and the circulating blood consists for a time mainly of primitive erythrocytes, a few macrophages and hemocytoblasts, and an occasional granulocyte. Then progressively larger numbers of definitive erythrocytes begin to circulate. The Wall of the yolk sac becomes thickened with dense masses. of spindle-shaped mesenchymal cells but apparently no real connective tissue fibers.
In the preceding pages the origin of blood cells in early human embryonic stages has been described on the basis of a graded series of excellently fixed and stained specimens.
The hematopoietic process is as follows: In primitive streak and somite stages some of the outstretched mesenchymal cells in the Wall of the yolk sac contract in places into rounded, deeply basophil cells, the hemocytoblasts. These multiply rapidly and give rise -to primitive erythroblasts, more numerous cytoblasts, a few macrophages and giant cells and, very rarely, granulocytes. In distinctly later stages (12 mm. and later) the hemocytoblasts give rise to small foci of definitive erythroblasts, more macrophages, and a few atypical megakaryocytes. All of these processes occur intravascularly. On a much more limited scale, primitive and definitive erythroblasts, a few granulocytes and a few phagocytes develop from the extravascular hemocytoblasts. Begirming with the 12-mm. stage, sporadic foci of definitive erythropoiesis appear in the entoderm.
Recent work gives us some definite evidence as to the origin of the mesenchyme of the yolk sac in primates. In the earliest implantation stages which have been obtained there are irregular strands of cells" traversing the relatively minute chorionic “cavity” (compare- Elder, Hartman and Heuser, ’38, and Heuser, ’40; also Hertig and Rock, ’39). These are the primitive mesodermal cells. As‘ ﬂuid accumulates within the blastocyst, these strands come ‘to lie on the available surfaces so that we have .a ehorionic, amniotic and vitelline mesenchyme as well as persistent magma strands and the more condensed cellular mass between embryonic and ehorionic vesicles which becomes the belly stalk (compare Streeter, ’26). In "the earliest stage of our series (H1496——ca. 13 days) the entodermic yolk sac vesicle is invested by an irregular network of_ mesenchymal cells (the extra-embryonic mesoderm of fig. H). As the embryo develops, the yolk sac becomes larger an its-mesoderm _tends to assume a more typi.eal epithelium-like arrangement (primitive streak stage). From the material available to us, the mesenchyme in the yolk sac wall, which will be the source of hematopoiesis, seems to be a derivative of this yolk sac mesoderm. A study of additional, adequately preserved human embryos of early primitive streak stages will probably settle this point. - In the mesenchyme of the chorion and belly stalk, the primitive vessels originate directly from the mesenchymal cells, apparently by simple transformation. These vessels at first are short disconnected structures, often with little or no lumen. In three instances ‘in our material We found abortive blood cell formation in vessels in the belly stalk. The many claims that these vessels are a frequent site of hematopoiesis are based on material which has not been promptly and adequately fixed, with the result that elective staining "of blood forming cells was impossible. Moreover, the groups of macrophages which occur frequently in the extra-embryonic mesenehyme have undoubtedly been interpreted as hematopoietic foci. It is only in the 18-somite embryo (about 24 days) in which the circulation is complete and functioning, that we have found immature blood cells in the chorionic vessels. However, these same cells also circulate in the vessels of the embryo proper. They undoubtedly continue to multiply and to mature in these vessels, but there seems to be little doubt that their site of the origin is the yolk sac.
The vessels in the yolk sac also arise from the mesenchymal cells, often as empty spaces similar to those in the chorion. But the picture here is complicated by the development of the so-called blood islands——the collections of hemocytoblasts which give rise to the blood cells. As discussed above, it is difficult in such situations to determine just what is the origin of the lining of the ves.sels. It is quite probable that in a group of hemocytoblasts (free mesenchymal cells) the peripheral ones and the out-stretched mesenchymal cells both contribute to the formation of the vessel walls, as described by Dantschakoff and Sabin for the chick- and Maximow for mammals. There is no evidence in this material for -the development of vessels through" transformation of individual hemocytoblasts.
We have no. convincing evidence of the development of vessel-forming cells from the cytotrophoblast as described by Hertig (’35). Indeed, we have only occasionally seen cells that might possibly be interpreted a transition stages between cytotrophoblast and mesenchyme. The endothelium of the early vessels in the yolk sac is not infrequently phagocytic and may occasionally break free as either a phagocyte or a hemocytoblast. The embryonic endothelial cells thus have the potencies of mesenchymal cells which happen to be in a particular location. “We think it best not to call them angioblasts; for the idea ,of angioblast to many embryologists implies that the cells which give rise to hematopoiesis and blood vessels are not simple mesenchymal cells but are "predetermined and must grow into an organ or region of the embryo after having-originated elsewhere.
It seems impossible on the basis of the material obtained until now to separate the hemocytoblasts into several distinct stem cells. The stem cells are of the same appearance in the various situations in which they occur irrespective of the various cytological methods used to identify them. It must be stressed that those who believe the stem cells to be of several types identify their specific stem cells, in actual practice, on the basis of the organ in which the cell is found or by the more mature cell types to which it-gives rise. Such a separation of stem cells is not made on morphological differences in the cells. Further, such attempts fail completely in ectopic hematopoiesis. The first hemocytoblasts (primitive blood cells of most authors) are not determined to be erythrocytes. They are true hemocytoblasts in that they give rise to a variety of blood cell types.
The claim that erythrocytes in mammals develop exclusively intra-vaseularly is not in agreement with our finding, for some primitive and definitive erythroblasts also develop extra-vaseularly. In the case of the latter generation, this occurs not only in the mesenchyme, but also within the entoderm.
The sharp separation of two generations of erythrocytes in this human material merits some comment. It seems clear now that, in all of the common laboratory mammals and in man, there are two distinct generations of erythrocytes, both arising from similar hemoeytoblasts "and developing through roughly parallel but morphologically distinct series of erythroblasts, that is, those of the primitive and definitive generations. In the mammals these are separated in time and space.
In the rabbit, guinea pig and cat, great numbers of definitive erythrocytes develop in the yolk sac, While in the rat and mouse definitive erythrocytes are not formed in this organ (Maximow, ’09; Jolly, ’23). In the human yolk sac, definitive erythrocytes develop, but their number is small compared with that in the yolk sac of the guinea pig, rabbit and cat.
A discussion of the relation of the large red blood cor puseles of pernicious anemia and their precursors to the primitive and definitive erythrocytes and erythroblasts is given by Jones (’38).
Hematopoiesis is described in a series of well-prepared young human ova starting with the 13-day stage (similar to the Peters ovum).
Except for a small focus in the belly stalk of the primitive streak stage and two foci in that of the 18-somite embryo early hematopoiesis is confined to the yolk sac. ' The hemocytoblasts are predominantly of large lymphocyte type, but-smaller examples corresponding to medium sized and small lymphocytes are found in the yolk sac vessels as early as the head process stage.
In the yolk sac, hemocytoblasts give rise to a. generation of primitive erythrocytes and to a subsequent independent lineage of definitive erythrocytes. The hemocytoblasts also give rise to macrophage, giant cells, atypical megakaryocytes, and myelocytes.
The primitive erythrocytes arise primarily intravascularly although a few are also found extra-vascularly.
The definitive erythrocytes develop within vessels, in the mesenchyme, and Within the entoderm.
Blood vessel primordia in the chorion plate, villi, yolk sac and belly stalk arise by direct transformation of mesenchymal cells. We have no convincing evidence of their direct development from cytotrophoblast. Except for three isolated abortive foci in the belly stalk, blood cells develop intravascularly only in the yolk sac vessels until at least mid-somite stages.
BLOQM, W. 1938 a. Lymphocytes and monocytes. Chapter V‘ of Downey’s Handbook of Hematology. New York.
1938 b Embryogenesis of mammalian blood. Chapter XIII of Downey ’s Handbook of Hematology. New York.
1938 c Tissue cultures of blood and blood—forming tissues. Chapter XX in D0wney’s Handbook of Hematology. New York.
BREWER, J. I. 1937 A normal human ovum in a. stage preceding the primitive streak (Edwards-Jones-Brewer ovum). Am. J. Anat., vol. 61, p. 429.
1938 A human embryo in the bilaminar blastodisc stage (The Edwards-Jones-Brewer ovum). Contrib. to Embryo1., vol. 27, pp. 85-93, from publication 496 of Carnegie Institution of Washington. 1940 In press.
BRYCE, T. 1904 The histology of the blood of the larva. of Lepidosiren paradoxa. Trans. Roy. Soc‘. Edinburgh, vol. 41, pp. 291 and 435.
DANTSGHAKOFF‘, W. 1908 Untersuchungen iiber die Entwicklung des Blutcs und Bindegewebes bei den Viigeln. I. Die erste Entstehung der Blutzellen beim Hiihnerembryo und der Dottersack als blutbildendes Organ. Anat. Hefte, Bd. 37, S. 471.
ELDER, J. H., C. G. 1-l.u':.'rMAN AND G. H. Hnnsnx. 1938' Ten and one-half day chimpanzee embryo, “Yerkes A.” J. A. M. A., vol. 111, p. 1156.
FERRATA, A. 1918 Le Emopatie. Soeieta Editrice Libiaria. Milan.
HERTIG, A. T. 1935 Angiogenesis in the early human chorion and in the primary placenta of the macaque monkey. Contrib. to Embryology, Carnegie Inst. Washington, no. 146, p. 37.
Hnerxe, A. T., AND J. C. B001: 1939 On a complete normal 12-day human ovum of the pre-villous stage. Anat. Rec., vol. 73, suppl. 2, p. 26.
HEUSER, O. H. 1932 A presornite human embryo with a definite chorda canal. Contrib. to Embryol., vol. 23, pp. 252-267. Carnegie Institution of Washington.
1940 The chimpanzee ovum in the early stages of implantation (about 10} days). J. Morph., vol. 66, p. 155.
JOLLY, J. 1923 T1-aité technique d’hematologie. Paris.
Joxns, H. 0., AND J. I. BREWER 1935 Normal human ovum in the primitive streak stage ‘(approximately 182; days). Surg. Gynec. and 0bstet., vol. 60, pp. 657-666.
Jomns, 0. P. 1938 Cytology of pathologic marrow cells with special reference to bone-marrow biopsies. Chapter XXVI. of Downey’s Handbook of Hematology. New York. ,
JORDAN, H. 1910 A microscopic study of the umbilical vesicle of a. 13-mm. -human embryo with pecial reference to the entoclermal tubules and the blood islands. Anat. Anz., Bd. 37, S. 12 and 56.
MAXIMOW, A. 1909 Untersuchungen iiber Blut und Bindegcwebe. I. Die friihesten Entwicklungsstadicn dcr Blut- und Bindegewebszellen. beim S5iugetierembryo, bis zum unfang dcr Blutbildung in der Leber. Arch. f. Mikrosk. Auat-., Bd. 73, S. 444.
1927 Bindcgewebe und blutbildende Gewebc. Handbch. d. mikr. Anat. cl. Menschen. (v. Miillendorff), Bd. 2, S. 232. Berlin.
RICE, A. 1%., M. M. Wrmnonn AND M. R. Lmwrs 1939 The" differentiation of myeloblasts from lymphoblasts by their manner of locomotion. Bull. Johns Hopkins Hosp., vol. 65, p.291.
SABIN, F. R. 1920 Studies on the origin of blood-vessels and of red blood corpuscles as seen in living blastoderms o£ chicks during the second day of incubation. Contrib. to Embryology, Carnegie Inst. Washington, vol. 1-1, p. 1. ‘
S.A.xEn, F. 1896 fiber die Entwicklung .und den Ban lder "not-malen Lymphdriisen and die Entstehung der roten und weissen Blutkiirperchen. Anat. Hefte, Bd. 6, S. 347.
STREETER, G. L. 1926 The “Miller” ovum—the youngest normal human embryo thus far known. Contrib. to Embryology, Carnegie Inst. Washington. vol. 18, p. 31.
Explanation of Plates
Plates 1 and 2 were drawn at a. magnification of 2240 X and rcdtlced to 1100. All of these preparations were stained in hematoxylin-eosin-azur II after Zenkerformol fixation. Plates 3 and 4 were drawn at 600 X and reduced to 400 X. figures H, I, .1’ were stained with hcmatoxylin and eosin, and figure K with hematoxylin-eosin-azur II, all after Zenker-formol fixation.
All plate were drawn by Miss E. Bohlman with the camera lucida.
Bnt., Entod., entoderm L,. lumen of early veel Sp1.me., splanchnic niesotlielium 1, hemoc-ytoblast 1’, the same in mitosis l.a,- transitions from hemocytoblast to primitive erythroblast 2, polychromatophil primitive erythroblust _ 2’, the same in mitosi 3, late polychromatophil primitive erythroblast ' 4, orthoehroma-tic primitive erythroblast (or primitive erythrocyte) 5, mcsenchymal cell 6, endothelium _ 7, polychromatophil definitive erythroblast 7‘, the same in mitosis 8, late polychromatophil definitive erythroblat 9, orthochromatic deﬂnitive erythroblast (normoblast)
10, histioid wandering cell (macrophage)
12, atypical megakaryocyte
13, eosinophil myelocyte
14, heterophil myelocyte
15, giant cell; perhaps an atypical primitive erythrocyte
16, phagocytosing hemocytoblat
17, partially cut definitive polychromntophil erythroblast
18, small macrophage, originating from a medium—sized hemocytob1ast
19, 20, medium-sized hemocytoblast with pseudopodia,
21, small ‘macrophage originating from a. medium-sized hemocytoblast
22, giant primitive polychromatophil erythroblast
23, small hemocytoblast,
24, macrophage intermediate between cells 18 or 21 and cell 11
25, tags in formation of binucleate primitive erythroblast
A Section of fold of yolk saw of H1516 (1.8 somites). The fold is covered with splanchnic mesothelium (Spl. mes.) and contains tortuous sinuses filled with early stages of primitive crythrocytopoicsis. The hcmocytoblasts (1) form a compact mass of cells; one is in mitosis. Cells 2, 3 are stages in the develop-ment of primitive polychromatic erythroblasts which end with cell 4, the primitive erythrocyte.
B Section of yolk sac wall of 20-mm. embryo (Maximow I19). In addition to the primitive erythrocytes (cells 4) there is.'intcnse production of polychromatophil definitive erythroblasts (cells 7, 7’, 8) leading to the formation of orthocln-omatic definitive crythoblasts (cell 9).
C This section is through a fold in the yolk sac of E1516, and like that of figure A, from the same yolk sac, is so sectioned as to be covered with splanchnic mesothelium. This is an earlier stage of the process than tl1.at shown in figure A. Here there are large numbers of extravascular hemocytohlasts (cell 1) of which one is in mitosis (cell 1’). There are also :1 few cells in the tip of a vessel. whose lumen is apparent at fl) Section of yolk sac of H1515 (head process stage). Between the entoderiu (Ent) and the splanchnic mcsothclium (Spl. mes.) are a. few meseiic-1ryrna.l cel.ls (cell 5) and four huomocytoblasts (cells 1). The cleft in the Yl'l(,‘H(-!IlL'.lJ._)’!Ile- does not ha.ve a. wall of its own and is not a vessel.
E Section of entoderm of yolk sac of 20-mm. embryo (Maximow H9) showing two entodermal cells, each with -.1 homoeytoblast.
F Section of entoderm of yolk sac of 20-nun. embryo (Maxiniow II9) showing ten early polyehromatophil definitive erytliroblnsts in an entodcrmzll cell.
G Section of yolk sac vessel of a slightly older embryo (“9 weeks,’ ’ ll/Ia.xi.1now H29). Note especially the macrophages (cell 11) with included green and yellow pigment.
(lells 12, 13, 14 are from the some yolk sac as those of figure. G. '1‘lIcy are, respectively, an early megakaryoeyte, an eosinophil rnyelocyl.-e, u. heterophil n'1yclocyte.
Cell 15 is from a. yolk sac vessel of the 1S—somite embryo (H1516).
Cells 16, 18 to 25 are from one field within a vessel. of the head process stage e.n1lrr'_)'0 (H1 51.5). The intervening cells, mostly primitive polychromutophil erythroblusts, have been cliinirlaterl to save space. These. cells are .identific(l in the list of ubbrex-'iati.ons. Note particularly that- cells 20 and 23 are typical n1ediun1—sized and small lymphocytes.
H Section through the embryonic disc and the yolk sac of embryo 111496 (stage of Peters’ ovum). 'l‘he extra-embryonic mesodcrm forms :1 network 0V-'61’ the surface of the cyst, lined with yolk sax-, elltnderm.
I Section of yolk sac wall (close to embryonic disc. of luzad process stage embryo H1455}. The 11¢.-l'.wo1k of mesodermal cells of figure l-1' has given rise to the mesenchyme and the splanchnjc nmsotlmlium shown here. The group of hemocytoblasts represents the first stage in hematopoiesis.
J This is from the n-b-embryonic portion of the sauna section of the yoik saw us figure I. Here hcmutopoiesis is muczh further advanced; the primitive vessels and mesexmhymal $_p11(‘A!H contain large areas of proliferating primitive erythroblums.
K Section of yolk sac of 20-mm. embryo (.\Ia.xim.ow I19). "Note 'Foci ul‘ dofiriitive c1'_vt.lu'oblz1sts in the entoderm and in :1. vessel.
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