Talk:Paper - Equivalence of different hematopoietic anlages 1 (1916)

From Embryology



The Wistar Institute of Anatomy and Biology


1. Introduction. Statement of the problems of hematology. Different

methods of attacking the problems. Myeloid metaplasis. Statement

of the problem of the present paper 255

2. Methods of investigation and terminology 267

3. Histogenesis in the spleen in relation to structural environment 274

A. Histogenesis in the normal chick spleen 274

B. Histogenesis in the chick spleen after stimulation 285

a. Changes after stimulation in early stages 287

h. Changes obtained by stimulation in the stage with definite

spleen vascularization 294

C. Conclusions. Data concerning: a) the histogenesis of spleen cells,

6) the conception of cell differentiation, histogenesis, c) the general meaning of myeloid metaplasis 296


Statement of the prohlerns of hematology. Different methods of attacking the problems. Myeloid metaplasis

The special hematological literature has been rapidly and constantly growing during the last decade, and there is enough ground to anticipate a further specialization in this partial domain of biology. Surveying the voluminous hematological literature, one is astonished by the discrepancy between the simplicity of the fundamental problems of hematology and the entanglement which they have undergone during the last




decade. One is naturally inclined to doubt, whether the small variations in the innumerable schemes are as important, as they are represented in the deductions of different workers.

The fundamental problems of hematology have a close bearing upon practical medicine as well as on the general problems of biology. Wilson and Conklin (6) especially, and many other biologists established as a fact the gradual segregation of different chemical materials collected in the egg. This is accomplished by a range of cleavages beginning in certain eggs at the first cleavage, in others delayed to later stages. As a result there appear groups of cells, different in their constitution and therefore possessing different potencies for differentiation. These cell groups proliferate and give tissues, which in their own differentiation may exhibit a great complexity. The diversity in the products of differentiation may be due either to differences in the physico-chemical constitution of the cells, or to differences in environmental conditions. One of the important questions of biology consists in determining the limit to which each of the agents cited has an active role. Does the segregation lead to a production of a definite number of uninterchangeable blood stem cells, of which the differences in the chemical structure imply a definite metabolism and their further specific development? Or does the process of segregation lead to a production of one group of numerous homogeneous primitive blood cells, which under different conditions in the embryo, as well as in the adult organism, split off variously differentiated cells? This problem was differently conceived by the monophyletic and polyphyletic schools.

The importance of a true conception of this fundamental problem of hematology for practical medicine is obvious'. The uninterrupted, everyday destruction of the blood elements is too often accompanied by a failure of regeneration in the organism. A whole range of stimulating agents for blood regeneration was found empirically. Still the success of interventions in similar cases as well as of interventions in various other deviations from the normal course of hematopoiesis (leukaemias) depends greatly upon a clear understanding of the action of the


agent on the processes underlying a definite symptom complex. An expenditure implies a complete knowledge of the reserve stock, the reserve being represented in the particular case by the group of cells, which by their uninterrupted reproduction and differentiation may supply the deficiencies.

The reserve stock is differently appraised by the monophyletic and polyphyletic schools. According to the polyphyletic school these reserves are formed by a number of specifically differentiated cell groups, which may multiply and ripen into still more specialized cells. The monophyletic school, on the contrary, finds in the organism along the various intermediate stages of differentiation also the young, undifferentiated polyvalent cells, which in the embryo have been the source of the various lines of blood cell differentiation. The monophyletic school admits a greater amplitude in the regeneration, and hence a wider sphere of influence upon the blood tissue. These common stem cells were identified by Pappenheim (31) as the large lymphocytes, by Dominici (10) as the small lymphocytes, by Weidenreich (44) and Downey (11, 12) as reticular cells.

However great the differences between the mono- and polyphyletic schools may seem, both groups of hematologists admit in the adult organism the existence of younger and riper cells and of their gradual differentiation products. The difference between them consists chiefly in the value of the amplitude of the differentiation process in the adult organism, in other words in the admission of different degrees of segregation, which may preserve or dissolve the common stem cells for various blood elements.

Various methods were applied for the elucidation of the fundamental problems of hematology, as well as for the understanding of the mutual relationship of the various .blood-cells and of the structure of the hematopoietic organs. As expected, the histological study of normal preparations of hematopoietic organs in adult organism was unable to solve the problem. The mere statement of coexisting cells in the hematopoietic organs offers a wide field for personal interpretation. Therefore the most


contradictory conclusions regarding the structure of the blood tissue were made on the basis of this method.

The embryo-genetic method may give more definite results if used exhaustively. The gradual appearance of the different blood cells becomes a reliable criterion for the judgment of the mutual relationship of the blood cells. The discovery of younger cells, which first develop in the embryo and the study of their gradual differentiation may be of great help in the identification of the blood cells in the more complex structure of the hematopoietic organs in the adult organism. However, the demand for strict exhaustiveness is often disregarded and leads to gaps, which are filled by a number of more or less keen interpretations of the investigator. The omission of the study of the first stages in the development of blood cells led investigators to an admission of specific stem cells for various differentiation products— Denys, (8), Bizzezero (3), and others. However, the recent histogenetic studies made by different investigators on various animals gave similar results. Bryce (2), Danchakoff (9), Maximow (25), Mollier (28), Haff .(18), all admitted that the different blood cells are derived from mesenchyme of mesodermal origin, and that the various blood-forming organs developed autochthonously at the expense of mesenchymal cells of which the various differentiation may depend upon external physico-chemical agents.

Studies of pathological changes in hematopoietic organs did not contribute greatly to the solution of the problems of hematology. The complexity in the mutual relationship of the blood cells was recognized and led many pathologists to take recourse to embryo-genetic studies, (Fischer (16,) Schridde (38), Schmidt (39) and others) . The conclusions drawn from studies made by these methods were based upon the existence of morphological similarities and differences between various blood cells. They lead to more or less plausible probabilities but do not determine irrefutably the functional dynamics of the various blood cells in different hematopoietic organs.


The recourse to the experimental method may contribute more definite information about mutual relationship of bloodcells as well as about the source of their regeneration and the function of different blood-forming organs. The study of intense general destruction of blood tissue through bleedings and of its partial destruction by X-ray application, followed by the study of its regeneration, have already contributed undeniable data. The use of a method known as lympho and myelotoxic intoxication, (I would better say stimulation) may give in this respect most conclusive results. This method was indicated in 1901 b}^ Dr. Flexner (17). Specific leucolytic, spleno- lymphomarrow-lytic sera are injected into animals. The consecutive circulation of antibodies present in these sera find adequate receptors in certain cell groups and a proliferation follows as a result of molecular changes in these cells.

One of the most striking results obtained in experimental pathology of the hematopoietic organs is the myeloid metaplasis of lymphoid tissue. First described by Fraenkel it was experimentally obtained by Dominici (10) by producing traumatic anaemias and typhoid infection. Later the myeloid metaplasis of the lymphoid tissue was repeatedly observed during various pathological processes, (anaemia, leukemia, intoxications, infections, tumors) . According to the statements of various investigators, the myeloid metaplasis invariably consists in a simultaneous development of erythro- and leucopoietic or granuloblastic tissues. This was accounted for by most of the pathologists as being a proof of a close relationship between these tissues. The common origin of leucocytes and erythrocytes was therefore admitted by many pathologists who formed the so-called dualistic school, which distinctly separated the myeloblastic (erythro-granuloblastic) tissue from the lymphatic tissue. The present paper has for its main subject the study of a very extensive chiefly granuloblastic (or myeloid) metaplasis of the embryonic mesenchyme, therefore a survey of different opinions concerning the subject may be permitted.

The myeloid metaplasis was observed chiefly in the spleen, and occasionally mentioned in the lymph-glands and other


organs of the adult organism. Dominici (10) admitted in his paper ('01) that cells with the structure of small lymphocytes give rise to the myeloid tissue. ^'Les petits mononucleaires en question (in another part of his paper he uses the term of small lymphocytes) ont des dimensions egales ou inferieures a celles des hematics : un noj^au rond, une bordure protoplasmique mince. Un element figure, offraiit de tels attributs, n'est ce pas ce que I'on a denomme communement une cellule embryonnaire?" A closer study of the embryogenesis of the blood tissue would certainly not have allowed Dominici to consider the small lymphocytes as being young embryonic cells, for the cells bearing the structure of small lymphocytes appear in the organism considerably later than other lymphatic cells, at least in mammals, birds and reptiles. Though the stem cells of the myeloid tissue, according to Dominici, bear a morphological structure identical with cells, which differentiate into lymphatic tissue, he still attributes to the myeloid stem cells specific potencies of differentiation, which they preserve from early embryonic period. Eater, in 1909, Dominici (10) changed his opinion and attributed to the lymphatic tissue itself the faculty of myeloid transformation. Dominici was thus first to stand for the conception of autochthonous development of the myeloid tissue in the lymphatic organs.

According to Ehrlich (13) the myeloid tissue, which appears under certain pathological conditions in different parts of the adult organism, derived from the central myeloid organ, namely from the bone-marrow. This opinion was expressed by Ehrlich at a time when the differentiation of every blood-cell seemed to be predestined generations back by the specific constitution of their ancestral cells. Ziegler (47) and Helly (19) still adhere to this view. They base their opinion upon the occurrence of bonemarrow Ausschwemnaungen, which follow venous injections of parenchymal mash, also after bone-marrow traumas. Helly finds support to his view also in the fact that after

Beeinflussung des Knochen-marks mittels Bakterien schon nach kurzer Zeit in der Milz des Kaninchens, welche unter normalen Verhaltnissen so gut wie gar keine specifischen Markelemente j lingerer


feneration wib etwa Mj^elozji^en, onthalt, eine derart hochgradige und herdweise auftrctcnde Einlagerung soldier Zellcn vorkommt, welche sieli iiieht nur ini Blute sondorn aiich in den Kapillaren anderer Organe reichlich finden, dass die Erklarung mit Hiilfe der gedachten Ver8chleppiing bei weitem die nahe liegendste ist.

However, the existence of myeloid metaplasis without any changes in the circulating blood, and further the discovery of diffuse autochthonous development of granuloblastic tissue in early stages of embryonic development, led most of the investigators to oppose strongly Ehrlich's conception of the myeloid metaplasis as being a metastasis from the central myeloid organ. If most of the investigators are inclined to consider the nweloid metaplasis as a local autochthonous process, their opinions concerning the source of the myeloid tissue, which develops in lymphatic organs, differ widely.

The fact that the myeloid metaplasis is chiefly localized in the pulpa of the spleen, became one of the main arguments of the dualists for denying any relationship between the lymphatic and the myeloid tissue in the adult organism. According to the dualists, cells from remote embryonic periods remained in the regions of the spleen-pulpa. Though their differentiation has not fully been accomplished, yet it reached stages at which these young cells respond to a stimulation by development of only myeloid tissue, (containing erythroblastic, leucoblastic tissue and megakaryocytes). According to them, the myeloid tissue, which in embryonic life is more diffusely spread out, does not disappear completely, but persists in a sort of latent stage. Bezangon et Labbey (1), Fischer (16), Heinecke (26), Lobenhofer (24), Meyer (27), Nageli (31), Schridde (38), Schmidt (39), Sternberg (40), Tiirck (42), and others uphold this view. Most of them attribute the development of myeloid tissue under pathological conditions to a "sudden wakening" of embryonic potencies in various cell-groups. These cell-groups are differently identified by different investigators. Preexisting myeloid tissue is the source of the myeloid metaplasis for Sternberg (40), indifferent lymphocyte like pulpa-cells for Meyer and Heinecke (25, 27), Schmidt (39), Lobenhofer (24), Fischer (16), connective


tissue cells for Fischer (16) and Klein (22) and finally reticulum cells for Klein (22) . iVn attempt was made to explain the myeloid metaplasis, not only by attributing potencies, characteristic for embryonic cells to cell-groups in adult hematopoietic organs, but also by assuming the existence of a mysterious process of dedifferentiation of already differentiated cells, Schridde (38), Fischer (16), Nageli (31).

Among the investigators who studied the myeloid metaplasis only a few admitted the existence of a common stem-cell for various blood-cells in the adult organism. Pappenheim (32), Werzberg (46), later Dominici (10) and Blumenthal (4) became supporters of the monophjdetic interpretation of the myeloid metaplasis. They pointed out that the localization of the new myeloid tissue during myeloid metaplasis is not as strict as the dualists admit. Moreover, the separate localization of the myeloid and the lymphatic tissues, where it exists, may become the differentiating factor for a common stem cell, which of course could not develop under equal conditions into different products. The monophjdetic interpretation of the myeloid metaplasis was corroborated to a great extent by embryogenetic studies, Bryce (2), Danchakoff (9), Maximow (25), Moliier (28), Haff (18) and by histological studies (Weidenreich (44), Downey (11, 12), Ferrata (33).

The study of myeloid metaplasis of lymphatic tissue has been in adult organs, in which at least a temporary embryonic or even a permanent partial granuloblastic differentiation existed. Therefore a possibility of persistence of specific granuloblastic stem cells in such organs could not be denied and a differentiation of specific cells could be explained by the specific constitution of their stem cells. Under definite stimulation, these specific stem cells might intensely proliferate. On the other hand, the hematopoietic organs could also preserve young specifically undifferentiated cells, polj^valent in their potencies. Their partial and local differentiation into myeloid tissue might have been caused by specific conditions of their environment.


It was repeatedly pointed out that the stem cells, which in the })ulpa and in the follicle of the spleen give different products of development, seem to bear a perfectly similar morphological structure, Dominici (10), Hirschfeld (21), Meyer u. Heinecke (26), Weidenreich u. Downey (12), Butterfield (5), and others. However, many recent data seem to indicate that the isomorphism does not imply either isogenesis nor especially isodynamics.

The study of myeloid metaplasis in such organs, in which both directions of differentiation coexist permanently in the adult organism or temporarily in the embryo, does not evidently offer favorable opportunity of solving the problem : whether the adult organism does preserve a stock of embryonic undifferentiated cells capable of various differentiation, or not. Neither do these studies solve the question: whether the morphological structure so characteristic of and common to the young cells both of the lymphatic and myeloid tissue is a result of definite physico-chemical constitution of which further changes are implied by differences in environmental conditions; or whether a group of morphologically identical cell units may have different physico-chemical constitution which would imply their further different development.

My (9) personal studies on the normal histogenesis of the ■ blood-cells and of the hematopoietic organs in birds and reptiles led me to a monogenetic conception of their origin. The study of regeneration of hematopoietic tissue after bleedings as well as that of changes undergone by this tissue during starvation (9) seemed to corroborate the monogenetic interpretation.

The admission of the existence of common stem cells in different hematopoietic organs implies therewith the admission of identical reaction of these stem cells to a stimulating agent as far as these stem cells are submitted to the same conditions. The structural environment in the full-grown organism is, however, highly differentiated in the various organs. The stem cells in different hematopoietic organs are of course expected to respond to stimulation by simultaneous proliferation; but their differentiation will be specific according to environmental conditions, for their differentiation depends upon conditions, usually


not experimentally controlled, upon the localization of the stem cell in the pulpa or in the follicle, in the case of the spleen. The possibility of identical reaction of the stem cells to definite stimuli is, however, not completely excluded. There may be certain kinds of stimuli, which may cause deviation from the normal differentiation of stem cells. A similarity of the reaction of stem cells in different organs may be expected also in case the different environmental conditions are made alike, or in case the environment is not fully differentiated, as for example in an embryo.

Under the influence of these premises an observation made by Dr. Murphy of the Rockefeller Institute attracted my attention. He observed 2-3 years ago an enlargement of the spleen in the host embryo after grafts of various tissues. ^ A closer study of this process led me to conclude, that the considerable enlargement of the embryonic spleen is induced by an intense proliferation of the young stem cells. This fact seemed to enable a

1 This observation was not published by Dr. Murphy at the time. While this paper was in press a brief note by Dr. Murphy regarding the general effect of . the spleen grafts on the organism of the host embryo appeared in the Journ. Exp. Med., July, 1916. It appeared after a number of. my papers and communications (Meeting at New Haven, Staff Meeting at the Rockefeller Institute) and after numerous demonstrations to Dr. Murphy of my preparations. Dr. Murphy states in the above quoted note, that at his suggestion I undertook "the working out of the finer histological details of the process," discovered by him. This, in view of the above mentioned facts, I venture to consider unwarranted.

Dr. Murphy writes the brief note for completeness and record," and gives a reference of his previous work, in which "observations were made on the effects of certain organ grafts on the embryo itself. Murphy, Jas. B., Journ. Exp. Med. 1913, vol. 17, p. 482." This paper however does not contain any observation on this subject. The only passage in this paper, referring to the effect of the graft upon the embryo, reads: "Apart from the thin continuation of the chick membrane, which covers the tumor and the ingrowth of vessels with their scant accompanying stroma, there is no histological evidence of reaction on the part of the embryo to the invasion of foreign tissue." Nor is it possible to find the slightest indication about the effect of the grafts in the body 'tissues of the host in any of Dr. Murphy's previous papers. Through personal communication from Dr. Murphy I knew about the enlargement of the embryo spleen. By deduction from my previous hematopoietic work I reached the conclusion regarding the necessary coexistence of analogous changes in other hematopoietic organs, a conclusion which the results of the experiments undertaken proved to be correct.


thorough revision of the fundamental problems of hematology. Indeed, if the various anlages of the hematopoietic organs were equivalent and contained idejitical stem cells, hematopoietic organs other than the spleen must have reacted also. Since the structural peculiarities in the different organs of the embryo are not fully differentiated, the reaction of the stem cells in different hematopoietic orga>ns could be expected to be more homogenous. As shown further, the specific intervention was indeed followed by changes both in the spleen and iji all the other hematopoietic organs and the reaction of these different organs was substantially similar. These facts were mentioned and demonstrated by me at the New Haven Meeting. ^

The grafting of adult spleen on the allantois of the embryo is a complex intervention. The grafted tissue contains small and large lymphocytes, together with the so-called reticular tissue, and with the vessels and their different layers, all belonging to a full-grown organism. Which of these elements has to be regarded as the source of the stimulation could not yet be defined conclusively. However, there is no doubt the intervention applied introduces in the embryo heterogeneous substances.

It is known that the organism reacts to the introduction of heterogeneous substances by a production of antibodies and to the introduction of heterogeneous cells by production of the so-called lysins. The erythrolysins which are developed by the immunized animal have a dissolving power on the red blood corpuscles against which the animal is immunized, the leuco ^Abstr. Proc. Anat. Record, Jan. 1916.

In this connection I desire to call attention to the substitution of a reference to Dr. Murphy's last communication (21) on p. 96 of No. 1, vol. 24, Journ. Exp. Med. July, 1916, in my paper on "Differentiation ..." for a footnote, in which I stated: "I wish to express my indebtedness to Dr. J. B. Murphy, who kindly demonstrated to me the method of grafting described in this paper. Murphy, J. B. and Rous, P., 1912. The behavior of chicksarcoma .... Journ. Exp. Med. vol. 15." My paper, which was received for publication in February '16, and published in July '16 could certainly not contain a reference to Dr. Murphy's communication, which received for publication in May, appeared also in July. This alteration seems to me the much the more inappropriate, because it has been made to apply to the demonstrations given by me at the Anatomical Meeting, 1915.


lysins stimulate the hematopoietic organs and induce intense proliferation of their cells.

Antibodies are regarded as being specific and may influence only cells in which they find adequate receptors. Dr. Flexner (17) has shown an apparent lack of specificity both of certain kinds of leucolytic sera to different hematopoietic organs and of different leucolytic sera to one definite hematopoietic organ. The spleno- lympho and marrow-lytic toxins, each of them acted in a stimulating manner upon all the hematopoietic organs, — hence the antibodies of the leucolytic sera evidently found cells with adequate receptors in all hematopoietic organs. These cells under the influence of certain amboceptors responded by common proliferation in different hematopoietic organs.

The results of the experiments cited offer a further corroboration of the monogenetic conception of blood development. The histological studies established in all the hematopoietic organs — the presence of cells, of which the morphological structure seemed to indicate a great potency for differentiation and proliferation. The embryogenetic studies pointed out these cells, as the true stem cells, common to all the hematopoietic organs and endowed with faculty to intense polyvalent differentiation. Finally, studies on stimulation of the hematopoietic organs by agents, which were supposed to be specific, seemed to indicate in all the hematopoietic organs the presence of cells, which respond to each of these stimuli by a common proliferation.

The experiments used in the present work are closely related to the experiments referred to above. The present study, however, is connected exclusively with the stage of antibody production. The requirement of heterogenicity of tissues might be found in the differences of the tissues in the adult organism and the embryo. (Even the morphological structure of the same kind of cells, for example the hemocytoblasts, changes somewhat with age, the cells undergoing an ontogenetic development.)

The intense proliferation, exhibited after the appearance in the embryo of heterogeneous substances by all the embryonic hematopoietic organs seems to indicate that different hemato


poietic organs in the embryo all contain elements, which react in analogous manner to the appearance of these substances. The fact that the proliferating cells exhibit a more homogeneous differentiation in the various embryonic organs than in adult organism may be accounted for by absence of specific structural environmental conditions in the embryonic organs. Though chiefly concerned in histogenesis of the blood cells, the present paper may have a close bearing on the immunity problem in establishing a connection between introduction of heterogeneous substances and reactions exhibited by hematopoietic tissue.

The grafting of an adult spleen on the allantois of the embryo produces changes similar to those described in myeloid metaplasis, and in principle are similar in different hematopoietic organs. However, these changes seem to be differently exhibited in hematopoietic organs at different stages of embryonic development. The stimulating agent may differently influence various cell groups in the hematopoietic organs at different stages of their development and leads often to the appearance of peculiar pathological processes. There appear in different organs characteristic changes depending upon their structural peculiarities which will be studied and described consecutively. As the most conspicuous phenomenon is the appearance of an enormous hypertrophy of the spleen, therefore a study of this organ will give a basis for further investigation of the changes undergone by other hematopoietic organs, including the mesenchyme of different parenchymal organs. A study of changes in the circulating blood of the embryo, as well as a study of the growth and differentiation of the grafts themselves will follow.


It has been shown in the introduction that the usual methods of investigations applied to the problem of the origin and the mutual relationship of blood cells failed in the attempt of solving them. There must be found new methods for the elucidation


of the problems so unsuccessfully discussed year after year. Such a new method for study of hematological and other biological problems may be offered by the study of transplantations of tissues on the allantois of an embryo and also in the study of the changes, which occur in the tissues of the host after grafting. The influence of identical environment upon different hematopoietic organs may be easily tested by this method. Transplantations on the allantois of embryos were used by Murphy and Rous (30) in their studies of transplantability of tissues to the embryo and by Murphy (29) in his study of the factors of resistance to heteroplastic tissue-grafting. Transplantations of adult spleen and bone-marrow seemed to supply the embryo with a refractory mechanism against heteroplastic grafting, which in a normal embryo is lacking. These transplantations as told are followed b}^ a considerable enlargement of the host spleen.

Every theory is a deduction of a limited number of facts, but if the theory is true, it must apply to all analogous cases. The enlargement of the embryonic spleen, mentioned above, which soon was discovered to be a true hypertrophy, could not be explained from the standpoint of the monophyletic school as an isolated process. The monophyletic school, if true, had to assume that changes in the embryonic spleen were accompanied by analogous changes in other hematopoietic organs. Since the hyperthrophy of the embryonic spleen has been involved by a considerable proliferation of the stem cells, stem cells in other hematopoietic organs admitted by the monophyletic school equal for all, must have been affected also and must have proliferated. Since the structural environmental conditions in the embrj^o are less differentiated than in the adult organism, the reactions in the different embryonic hematopoietic organs may be expected to be more homogeneous. As will be seen later, this assumption, resulting from the premises of the monogenetic conception of the blood origin, has been fully confirmed by the results of the experiments.

In principle similar to the tissue cultures the grafting method has a. great advantage over them. The allantois offers for the


transplanted tissue ideal conditions in supplying the growing tissue \^^th nutritive material and also in withdrawing the products of the metabolism of the developing and differentiating cells. Though the tissue growth and differentiation cannot be observed right under the microscope, this is compensated by the facility of obtaining abundant experimental material in dilTerent development stages.

As Murphy and Rous (30), I used the method developed by Peebles for studies in experimental embryology. Since I had to overcome many difficulties, and failures were due sometimes to apparently minute details, I will undertake a thorough description of the culture-method on the allantois of the chick embryo, as I used it in my experiments. I am indebted to the kind efforts of Mr. Ebeling of the Rockefeller Institute for being supplied through the whole winter with fertilized eggs, which developed in the incubator in the number of 60 to 70 per cent. The cultures succeed on the allantois easily from the beginning of the 7th day of incubation. The cultures of the hematopoietic organs grow if in contact with the mesodermal surface of. the allantois. The ectoderm, which overlies the area vasculosa and the 3'olk does not offer favorable conditions for cultures of hematopoietic tissue. From the 7th day of incubation, however, the allantois appears as a well-sized sac, flattened under the eggshell.

It is easy to determine by means of illumination, which of the incubated eggs started to develop. The localization of the embryo body as well as that of the allantois with its vessels comes therebj^ clearly out. Good places to choose for grafting are regions between the junction of two vessels at a distance of 1 to 2 cm. £rom the embryo body. The illumination and the provisional marking of the eggs must be done quickly and the eggs immediately returned to the incubator. A sawing out of small windows in the region marked on the eggshell follows. It is advisable to saw the windows in the form of a trapezium, which form allows to orientate it easily, when the window has to be closed. A great care must be observed in sawing the windows out of the eggshell. The pressure of the instrument has


to be slight, otherwise the shell cracks easily outside the region marked. Splits may be stopped by paraffine and the egg still used. As instruments for sawing, small scalpels used in ophtalmologie may be recommended, a few jaggs on their edge are often useful. An experienced hand will easily determine the time when the eggshell is passed through. Then the egg is again returned to the incubator. After all the eggs are opened instruments for aseptic extraction of organs, (a few scalpels, scissors, forceps and bone cutter), and instruments for grafting, (tissue crusher — it is a syringe with a bolt-bottom, another syringe with divisions of 0.1 gram, and a needle, a fine forceps and two pairs of scissors), are sterilized.

All the next work has to be done quickly. The organ, aseptically extracted, is cut in small pieces and passed through the tissue crusher, the tissue mash is then pulled into the syringe and is ready for grafting. Now egg after egg is taken out of the incubator, the shell window is removed, the shell membrane is lifted by the small forceps and an opening cut out with scissors. The allantois becomes visible and often in earlier stages falls off somewhat from the eggshell. Through the needle 0.1 to 0.2 of the tissue is then pushed in, introducing the tissue if possible under the eggshell at least under the shell membrane. Great care must be taken in grafting eggs in advanced stages, because the allantois then bleeds intensely, and the contact of the transplanted tissue with the allantois is removed by the extravasat. No graft usually takes, if the grafted tissue is introduced into the cavity of the allantois or deeper. In these cases the tissue is found floating in the form of a greyish mass, containing numerous small grains.

After the tissue has been implanted on the surface of the allantois, the window is closed by the piece of the eggshell withdrawn and the sphts are covered by paraffine. The paraffine of a higher melting degree is preferable, in order to prevent its melting during the following incubation of the eggs. If a local graft is desired any mechanical disturbance should be avoided, otherwise a more diffuse distribution of the grafted tissue is easily obtained. The graft takes more successfully if the egg


during the first twelve hours is put in the incubator, the window down. This secures a closer contact of the tissue introduced with the surface of the allantois. Next day I usually changed the position of the egg in directing the window above. The strict observance of the directions given secures usually 80 per cent of successful cultures. Interesting results in the form of diffuse growth of transplanted tissue were obtained by introducing an emulsion of the tissue mash between the allantois and the chorion under the eggshell. The whole allantois appeared in a few days covered by innumerable small grafts. The same results were occasionally observed also after applying the usual method of grafting.

Tw^elve hours after transplantation, the tissue is usually in firm contact with the allantois. Later it is attached by nunierous vessels growing from the allantois into the tissue. For fixation of the material, it is advisable to free and fix first the embryo after a ligature of the vasa umbilicales is done ; then to cut out a large piece of the shell in the region of the culture. The allantois which covers the shell is transported in the fixing fluid together with the shell and is removed from the shell 5 to 10 minutes later. The fixation of the allantois and of the graft is completed in ^ to 1 hour in Zenk.- formol. The celloidin was successfully substituted by the parloidin Dupont, and I may recommend this product as being in no way inferior to the celloidin Schering. The imbedding in celloidin or parloidin remains a sine qua non for hematological work, what easily was deduced by a study of a few specimens imbedded in paraffine last autumn (1915) when no more celloidin was available. Since the staining of preparations attached to the slides and freed from parloidin is more effective, it may be permitted to recall the method of Rubashkin (36), somewhat modified by Danchakoff (9, '08d).

The greatest part of the material was stained by eosin-azur and some of the preparations by Domini ci and Pappenheim. For the staining of fibrous tissue the iron-hematoxylin and subsequently van Gieson were used. Most of the illustrations are given in black on account of the special temporary conditions,



and one colored plate is added, as example of the preparations, from which the ink drawings were made. The photographs on plates 1 and 2 were kindly made by Mr. Schmidt of the Illustration Department of the Rockefeller Institute.

Since the nomenclature in the hematology has become nowadays extremely complex and often under one name different cell units are understood, or even oftener one cell unit is termed by different names, it is useful to state in advance what terminology will be used in this paper.

The stem cells of the different blood elements, which first appear after isolation of the blood-islands, have the structure of the well-known large lymphocytes. The term of large lymphocytes was appUed for these cells by Pappenheim (33), Danchakoff (9) and Maximow (25). Studies of hematopioetic tissue led to recognize everywhere cells of the structure of large lymphocytes and to identify them as stem cells for the blood tissue. Only few differentiation potencies were first assigned to the large lymphocytes. This however corresponds but little to the various differentiation potencies, exhibited under different conditions by this cell. So the name of large lymphocyte seemed to correspond little to the given cell in its new conception. Since personal studies did not give me any data, bestowing all the lymphatic cells with equal potencies, the less appropriate seemed to me the name of large lymphocyte in connection with the stem cell for different blood elements. In my last papers I called the stem cell, which in itself is a good name, lymphoid hemocytoblast — lymphoid in order to take into consideration its morphological structure, hemocytoblast on account of its potencies to differentiate into various blood cells. The same name will be used throughout the paper. The names of erythroblasts and erythrocytes do not require any explanation.

The names of myeloblast and myelocyte seem to me unfitted for the purpose used. This name is wrongly applied to cells which neither appear first in the bone-marrow, nor are cell units exclusively characteristic of this organ at any time of its existence. These terms will be substituted, as in my previous papers, by granulocytoblasts and granulocytes or leukocytes.


However, the term myeloid tissue, or myeloid metaplasis, is used throughout the paper as a collective name, under which all the characteristic cell elements of the bone-marrow are understood, it is the erythroblastic, granuloblastic tissues, and respectively the megakaryocytes. The terms promyelocytes, metamyelocytes, mikromyelocytes, which correspond to intermediate stages between a lymphoid hemocytoblast and a leukocyte are omitted. These stages are characterized by unessential features and are often overstepped in embryonic life during intensive regeneration. The use of so many terms for expressing small differences between development stages in a cell lineage seems more to confuse than to help. For this reason I did not introduce them lately in the scheme given in the Anatomical Record, and in the present paper, only terms which designate definite morphologically well defined stages will be used, and these are lymphoid hemocytoblast, granulocytoblast, and granulocyte or leucocyte.

The reciprocal relations in the lymphatic cell group are somewhat more obscure. The different lymphatic cells are looked upon by Maximow (25) and Weidenreich (44) as being merely temporary appearances of young undifferentiated cells, all characterized by the same differentiation potentialities. Though these authors admit a specific morphological structure for the large and the small lymphocytes and the histogene wander cell, yet they assume that these cells may easily change reciprocally their structure according to the environmental conditions. In birds and reptiles (Danchakoff (9)) as well as in mammals (Maximow (25)) it is easily demonstrated that small lymphocytes may both proliferate and differentiate further, but their lines and products of differentiation are not identical with those of the large lymphocytes (Pappenheim (32), Danchakoff (9) ). Neither is it proved definitely that the small lymphocytes may grow into the large. Nor is a possibility of erythrocyte development at the expense of small lymphocytes shown to exist in birds and reptiles, as Freidsohn (14) admits lately for amphibians and Venzlaff (43) for birds. Therefore, under the name of small lymphocyte, cells characterized both by a definite


morphological structure and by well defined differentiation potencies will be understood.

The histogene wander .cells, which term I substituted by histiotopic wander cells seem to be in close relation to the hemocytoblasts and often are merely an intermediate but morphologically well defined stage of development between a mesenchymal cell and a hemocytoblast.

I wish to express my thanks to the director of The Wistar Institute of Anatomy and Biology, Dr. M. J. Greenman, and to the Staff of the Institute, for the generous hospitaUty shown to me. I am indebted to Dr. S. Flexner for the kind admission to the laboratories of the Rockefeller Institute for Medical Research where the work has been partly done; to Dr. C. E. McClung for the great interest shown in my work and for the revision of a number of my preparations; to Dr. F. P. Mall for the revision of the text and the proofs during my absence.



The study of histogenesis in the spleen by the method of stimulation of the stem cells in the spleen anlage requires a thorough knowledge of the histogenetic processes, which normally take place in the spleen. The early stages of spleen development in birds were studied by Tonkoff (41). Yet no investigation of embryogenesis of the characteristic spleen elements was made by modern technique. The general lines of differentiation of the spleen elements were studied by Danchakoff ('16a) in Tropidonotus natrix, but the question — what are the conditions which determine the differentiation of the polyvalent stem cell — remained unsolved. It seems, therefore, necessary to make first a study of normal spleen development in the chick, and to attempt to deterixdne the conditions w^hich imply the various differentiation of the stem cells.

The appearance of the spleen anlage in the chick embryo and its first development corresponds to the description given by


Tonkoff (41) in 1900. The spleen anlage appears in an embryo nearly 4 days old — in the mesenterium dorsale duodeni in the region of pancreas dorsale. It is distinctly separated from the coelomic epithelium, which surrounds it. In the I stage of its appearance the spleen anlage is purely mesenchymal. It is distinguished from the surrounding mesenchymal tissue by the denser appearance of the tissue. At this time mesenchymal cells of the anlage present short ramifications, which soon are lost, the cells multiplying intensely and joining finally in a common syncytium (fig. 5 and 6). The spleen anlage at early stages is identified rather by its localization than by the character of its cells. Similar agglomerations of syncytium-like mesenchyme are encountered in many other places, and their further development exhibits a great analogy with that of the spleen anlage, resulting in a differentiation into lymphatic tissue. The more intense proHferation of the mesenchyme in the region of the spleen is evidently due to local favorable conditions. The development of the spleen at the expense of mesenchymal cells without any relation to the endoderm nor to the coelomic epithelium may be regarded as a well-founded fact.

The appearance in the spleen anlage of numerous ameboid cells, the lymphoid hemacytoblasts, and their differentiation into granulocytoblasts and into small lymphocytes was described by Danchakoff (9) Cl6a), in Tropidonotus natrix. The fact that the small lymphocytes develop in the spleen in later stages was also noticed. However, it is improbable that the different stages in themselves should be accounted for as differentiating factors. If the small lymphocytes do appear later, the granulocytoblasts nevertheless continue to differentiate in the spleen, at least in embryonic life. Conditions for both lines of differentiation must therefore coexist in later stages. The differentiating factors should be sought rather in different structural conditions, appearing at definite stages, and determining from the time of their appearance the lines of differentiation of the polyvalent stem cells.

Since the structural peculiarities are exhibited in a more striking manner in the spleen of an adult or a young chick, it


may be advantageous to demonstrate them by a study of a fully developed spleen, and then to attempt to find out whether the gradual development of the peculiarities influence the histogenesis of the hematopoietic tissue. A distinctive feature in the spleen structure is given by its special vascularization. The regions with veinous and arterial vascularization, though they penetrate each other, remain nevertheless independent, and communicate together only in places, where the white pulpa passes into the red. The most characteristic cell element of the spleen — the small lymphocyte — belongs to the white pulpa and accumulates here in the form of follicles and follicular strings. The granulocytoblasts, though not numerous in the adult spleen, are chiefly localized in the pulpa. Other ameboid elements, the basophilic large lymphocytes (lymphoid hemacytoblasts), and mononuclear leucocytes, often in the form of macrophages, are common to all the regions of the spleen. The syncytial cell reticulum is also ubiquitous; it forms in the red pulpa wide meshes. In the white pulpa the cells of the reticulum appear denser, and the meshes formed by their ramifications are smaller.

As a further study of the spleen development will show, the chief characteristic feature, primarily, determining different regions of the spleen as red or white pulpa, consists in the type of vessels by which a region of the spleen is supplied rather than by the presence of certain kind of ameboid cells. The wide veinous capillary net together with sinuses and lacunae forms the red pulpa, the bunches of arteries resolving themselves into a net of narrower branches — belong to the white pulpa. The question, whether the specific differentiation of the ameboid elements depends upon the peculiar vascularization of the spleen may be decided after a study of the spleen development. The chick spleen is a favorable subject for elucidation of this question, for the identification of the vessels is easy in the spleen anlage from the time of their appearance.

As mentioned above, the development of the spleen in the earliest stages is characterized by its loose mesenchymal structure. The intense cell proliferation leads soon to a transformation of the loose mesenchymal anlage into a denser syncytium.


Numerous basophilic cells differentiate at the expense of the syncj^tium and become ameboid (figs. 5 and 6, L. Hhl.). Acidophylic granules appear in the cytox:>lasm of a part of these cells and characterize them as granulocytoblasts (fig. 8, Grbl.). The development of these cells (lymphoid hemocytoblasts and granulocytoblasts) is not specific for the spleen mesenchyme and is observed in other regions of the embryo body also. At the same time the spleen anlage becomes vascularized, and its vascularization is at this stage of its development exclusively veinous. In the peripheral layers of the anlage, later on through the whole organ, appear splits, which evidently are filled by a liquid, which separates the cells. These splits are at first surrounded by the irregular surface of the mesenchymal cells. Some of the cells show still their processes, projected in the lumen of the sinuses, (fig. 6, S.). These sinuses soon join together and form a net. From the other hand a communication with branches of the intestinal veins is established. The whole mesenchymal anlage exhibits at this second stage of its development a spongious structure. Whether the appearance of the splits in the anlage is due to a secretion of the surroimding cells, or to a transudation of a liquid through the vessels growing from outside is difficult to determine. There is, however, no doubt that the splits mentioned are of local origin. This has been shown by Laguesse (23) in fishes.

The appearance of the splits in the tissue of the spleen anlage is accompanied by more intense isolation of ameboid cells (fig. 6, L.Hhl"). Some of them are surrounded by a developing sinus and become situated in its lumen. These cells have invariably first the structure of lymphoid hemocytoblasts (large lymphocytes). Similar cell groups are not seldom encountered in the larger sinuses of the peripheral layers in the spleen, (fig. 5,

  • S). As soon, however, as these lacunae unite with the veinous

vessels, what is indicated by the sudden appearance of differentiated erythrocytes within the lacunae, the lymphoid hemocytoblasts begin here their differentiation into erythroblasts (fig. 5, Erhl.). The plasma of the blood must evidently contain factors for differentiation of lymphoid hemocytoblasts into ery


throcytes. The slowness of the blood current in the large veinous capillaries and sinuses offers moreover favorable conditions for this line of differentiation.

The vascularization of a normal spleen proceeds, however, gradually. The cells, surrounding the splits, become gradually flattened and finally form an even endothelial surface (fig. 8). The sinuses of a normal spleen contain usually merely a few young cells undergoing an erythroblastic differentiation. I do not think it right therefore to consider the normal embryonic chick spleen as an active erythropoietic organ, though potentially it must be considered as such. It may be noticed that spleens of embryos at the same stage may offer in this respect well pronounced individual differences. New sinuses continue to appear with the growth of the spleen. In later stages, at the Uth day of incubation, as the fig. 7 shows, in newly formed sinuses there may be . found large groups of lymphoid hemocytoblasts, which soon undergo an erythroblastic differentiation. Between the new formed vessels the mesenchyme continues to proliferate and to split off lymphoid hemocytoblasts. They multiply also and partly differentiate into granulocytoblasts, which increase also their number by mitosis and partly differentiate further into granulocytes (figs. 8 and 9) .

The processes of growth and differentiation proceed slowly in a normal spleen. In an embryo of 9 days — 5 days after the beginning of its development — the size of the spleen reaches 1— 1.5mm the long diameter. The abundance of nuclei however may serve as index of intense proliferation processes taking place in the syncytial tissue of the embryonic spleen. The spleen during its II stage of development is characterized by a development of a net of wide veinous capillaries developed in the mesenchymal syncytium,, by an intense granulopoiesis outside the vessels and by a potential erythropoiesis within the vessels. These lines of differentiation, as known, are also characteristic of the myeloid metaplasis in the spleen pulpa. The study of the normal spleen development leads to the conclusion that the first differentiation processes of the mesenchymal spleen anlage transform it into a pulpa-like organ (figs. 7 and 8). The spleen remains pulpa-like until the 12 to 13 day.


The intense development of the arterial vascularization begins at this time and the spleen enters in the III phase of its development, characterized by the appearance of follicles. A cursory glance on spleens during development of arteries brings forth a striking difference between the development of the veinous and of the arterial vascularization. The arteries and their smaller branches never appear as irregular splits limited by mesenchymal cells. These vessels on the contrary always develop as regular narrow tubes (fig. 9, Art.c.) I did not intend to undertake a special study of the vascularization of the spleen, therefore I did not apply special methods of investigation for this purpose. However, a thorough study of preparations may give some information upon the development of the veinous and arterial vascularization. The veinous sinuses and capillaries are local formations, the arteries and their branches seem to grow into the spleen from outside and here ramify by budding (fig. 9).

The arteries, growing into the pulpa like tissue of the spleen, divide it in regions, which are soon further subdivided by smaller arterial ramifications. The arteries lie first in the mesenchyme, where numerous granulocytoblasts are present (fig. 9). The mesenchymal cells continue to proliferate around the arteries. The process of splitting off of lymphoid hemocytoblasts persists in these regions, but their differentiation into granulocytoblasts is suspended under conditions in which the mesenchymal cells develop around the narrow arteries. These conditions do not correspond to those which prevail around the thin walled veinous sinuses. The arteries and their smaller branches finally become surrounded by clear zones of mesenchymal cells (fig. 10), which markedly contrast with the granuloblastic tissue. These zones appear in preparations in the form of islands of mesenchymal tissue, which fill up all the interstices between the arterial vessels and the pulpa like tissue of the spleen anlage. These islands are anlages of follicles.

At the time of the intense development of the mesenchyme around the arteries a new line of cell differentiation may be traced in the spleen which soon will become predominant; this is the differentiation of small lymphoctyes. The figures 12


and 13 show the development of small lymphocytes in the follicles. From right to left the figure 12 represents the tissue of the follicle from the periphery to its center. In the peripheral parts of the follicle an intense isolation and proliferation of lymphoid hemocytoblasts takes place (5 mitoses in a part of the microscopical field) which leads to the formation of dwarf hemocytoblasts (fig. 12 S.L.Hbl.). A process of differentiation, starting in the groups of small hemocytoblasts (fig. 12) soon transforms them into true small lymphocytes (S.Lmc). The cytoplasm around the nucleus of these cells becomes smaller, the typical nucleolus of the hemocytoblast is replaced by chromatin particles which may be now permanently discovered in the nucleus (fig. 12, S.Lmc). A similar development of small lymphocytes is show^n in the figure 13 in the region adjacent to the red pulpa (right edge of the drawing). Both in the red and in the white pulpa numerous larger and smaller hemocytoblasts are present and offer in both regions similar morphological structures. In both regions they sometimes appear as groups of cells joined together, — probably an index of their syncytial origin. The further differentiation of the lymphoid hemocytoblasts is shown to be different, according to their localization. In the red pulpa they are in close contact with the larger veinous capillaries and differentiate into granulocytoblasts. In regions with scarce arterial vascularization they undergo a differentiation in small lymphocytes.

As in the thymus, the differentiation of small lymphocytes is preceded in the spleen also by development of generations of small-sized hemocytoblasts. Their appearance in the thymus seemed to depend upon an intense proliferation of the cells in a limited space. If the cells in the spleen are not heaped up, as they are in the thymus, yet the analogy of the conditions for their differentiation in both regions may be easily traced. Both in the thymus and in the spleen the small lymphocytes develop under conditions of poor nutrition. They appear in regions of the spleen where the swift blood current passes by the narrow arterious vessels.

The first groups of small lymphocytes differentiate between


the 15 to 17 days of incubation. They increase their number by differentiation at the expense of lymphoid hemocytoblasts and by their own proliferation. They gradually infiltrate the regions with arterial vascularization; finally they accumulate in dense masses around the arteries. One may observe amongst them a few degenerated cells, even in an embryonic spleen; they are phagocytosed by mesenchymal and endothelial cells and sometimes even by hemocytoblasts. A number of mesenchymal and endothelial cells are gradually transformed into typical macrophages (Evans) (15). Similar macrophages are observed also in the lumen of the sinuses, where they lie free, or form a part of the surrounding mesenchymal tissue (fig. 17, 18, 19), In the pulpa, however, the activity of the macrophages is directed chiefly toward the degenerated erythrocytes. Though at this time the connection between the arterial and the veinous vessels is completed, these regions continue to be very distinct.

The appearance of the follicles and of the new line of cell differentiation does not seem to influence the life of the cells in the pulpa. Both the granuloblastic and the lymphoblastic processes of differentiation coexist now in the spleen, and are displayed by lymphoid hemocytoblasts in different regions under different structural conditions. The granulo- or leukopoiesis develops around the large veinous sinuses under conditions identical to those under which they develop in the yolk sac and in the bonemarrow. The leukopoiesis in the spleen is much reduced after the embryo is hatched, and the spleen becomes chiefly a lymphopoietic and erythrolytic organ.

The structural peculiarities which determined the various lines of differentiation of the polyvalent stem cells remain in the adult spleen unchanged. Different stimulating agents may cause a proliferation of the stem cells. Their differentiation, however, will correspond to the structural environmental conditions to which they are submitted. It is therefore only natural that the myeloid metaplasis in the adult spleen should develop in the pulpa, where the structural conditions determine normally a granuloblastic differentiation of the hemocytoblast. A differentiation of small lymphocytes in the pulpa, or vice


versa, could be expected only in the case if the structural conditions of the pulpa be changed and corresponded to those in which the small lymphocytes normally develop.

The study of the normal development of the spleen in the chick allows the following conclusions :

1. The anlage of the spleen develops from the mesenchyme of mesodermal origin. It appears at the end of the 4th day of incubation. It consists first of a loose mesenchymal tissue, which gradually becomes denser and finally is transformed into a syncytium.

2. The development of a veinous vascularization transforms the spleen anlage into a homogeneous pulpa-like tissue. This stage is characterized bj^ a well developed granulopoiesis and a potential erythropoiesis. The stem cell for both directions of differentiation is the lymphoid hemocytoblast, which develops at the expense of mesenchymal cells. The line of the stemcell differentiation depends upon the conditions to which the stemcells are submitted.

3. The development of the follicles and of the lymphoid tissue coincides with the development of the arterial vascularization. The small lymphocytes develop at the expense of the lymphoid hemocytoblasts and their differentiation is preceded by appearance of generations of small-sized hemocytoblasts. The small lymphocytes in the spleen, as well as in the thymus, appear in regions, characterized by special structural conditions of the environment.

Different kinds of cells appear, as final results of differentiation processes, observed in the spleen. Reticular tissue cells, and endothelial cells, erythrocytes, granulocytes, small lymphocytes and macrophages develop in the same organ. Can anything definite be told on the basis of the study of normal spleen development about the origin of these cells? Are all the cells of the mesenchymal spleen anlage identical and polyvalent, their further development being determined by the structural environmental conditions, or though apparently identical in morphological structure their potencies to different development are predetermined by intrinsic imperceptible differences between them?

The general outlines of the development of the spleen anlage


under normal conditions should be regarded as necessarily determined. The organ develops in a complex environment of other organs which proliferate and differentiate at the same time. Each of these factors, which necessarily ensues from the collective action of all the surrounding conditions, becomes itself one of the factors which partly impels the results of the general differentiation. The necessity and mutual dependence of the development processes result from reciprocal influence of the factors and is characterized by striking purposefulness. The general outlines of the development may therefore be changed, as will be shown later, only in the case if the regular interaction of factors has been disturbed.

Variations in the intensity of differentiation of various cell groups may be observed, however, in normal spleens. Numerous or merely scant lympho- and granulocytoblasts may develop at the expense of mesenchymal cells. The proUferation of granulocytoblasts may also be more or less intensive. But this differentiation process is extended in the spleen anlage more or less uniformly at a time when the anlage presents a homogeneous structure and is lacking special conditions of vascularization. The differences in the extent of the normal granulopoiesis are, however, not excessive and deviation in other lines of differentiation may compensate them. A new line of differentiation necessarily starts with the appearance of new structural characters, namely with the development of the arteries. After this new factor has been ' established, it will permanently influence the cells, which stand under its control. The existence of regular unchangeable relations between definite structural conditions and differentiation of certain kind of cells seems to explain sufficiently the necessity which appears in a group of identical stem cells to diverge in their further development. Is there a need of recurring to invisible differences between cells, lest it should be necessarily required by deduction from the results of development?

One phase in the spleen development could, however, be interpreted by the dualists in their favor— this is the apparently late ingrowth of the arteries in the pulpa-like spleen anlage. If both


arteries as well as veins developed in loco, there would remain no doubt in the common origin of the lymphatic and the myeloid tissues. The ingrowth of the arteries may be regarded as a mere stimulus for a new intense proliferation of the mesenchyme and its subsequent differentiation into small lymphocytes. On the other hand, the ingrowing vessels might have brought new cell material for a new Une of differentiation, which starts at this period. It is difficult to establish with certainty whether the mesenchymal cells of the follicle anlages develop at the expense of the local mesenchyme, which remains undifferentiated, or whether they are derived from the tissue brought by the growing arteries. No matter how this question will be solved, both in the pulpa and in the follicles, the mesenchymal cells bear the same morphological structure. They present moreover in their first differentiation stages undeniable analogies. In the pulpa as well as in the folhcles they partly differentiate into lymphoid hemocytoblasts and partly form the so-called reticular tissue. The special differentiation according to environmental conditions is then undergone by the lymphoid hemocytoblasts.

The dualists admit that the development of the myeloid metaplasis of the spleen, which under definite pathological conditions is localized in the pulpa is due to proliferation and differentiation of adventitial or endothelial vascular cells. Endothelial cells are present both in the pulpa and in the follicles, the adventitial cells are even more numerous in the arteries of the follicles. If the strain in the fm-ther differentiation of the lymphoid hemocytoblast has to be laid in the cell itself and not in the physico-chemical conditions, given by structural peculiarities of the environment, the myeloid metaplasis should have appeared as a diffuse process, for endothelial and adventitial cells are found in the pulpa as well as in the follicles. In order to remain consequential, the dualists ought to recognize various kinds of adventitial and endothelial cells — the ones being capable of a myeloid differentiation, the others not.

The striking dependence of the various differentiation of morphologically identical cells upon a change in the environmental conditions may greatly strengthen the monogenetic


conception of the blood origin. It cannot, however, be considered as definite proof, because various differentiation products could under different conditions derive from isomorphic but heteropotential cells. The study of the spleen development under influence of stimulation of its stem cells will give further information about causal relation between structural environmental conditions and development of the polyvalent stemcells.


An intense stimulation of the mesenchymal spleen anlage is obtained by means of grafting a tissue mesh of an adult spleen on the allantois of a chick embryo. The photographs on figures 1 and 2 demonstrate the enlargement of the host spleen. Both photographs belong to an embryo of 18 days. The spleen is enlarged in both embryos. Wliile the spleen on figure 1 as compared with a normal is enlarged approximately 4 times the spleen on figure 2 shows a much more intense enlargement. The long diameter of the spleen at this stage is about 1-2 mm. After the stimulation it may exceed 1 cm. It is remarkable that the enlargement of the spleen is usually in a visible relation to the intensity of the graft growth. The spleen a corresponds to the graft on figure 3, the spleen b to the graft on figure 4.

The macroscopical appearance of the enlarged spleen does not give any indication whether the spleen enlargement is due to a local proliferation of the embryonic tissue. This proliferation could be incited by heterogeneous products of metabolism, which from the transplanted and growing cells could penetrate in the vessels and be transported by the blood current into the embryo body. On the other hand, cells from the graft could be transported in the spleen and here under favorable conditions proliferate. Tumor like accumulations of tissue would develop in the last instance and enlarge the spleen. The surface of the spleen after stimulation is often covered by protuberancies, numerous small whitish points may be seen on its section. These facts at first suggested rather the idea that the enlargement of the spleen was caused by tumor-like metastases brought by the blood


current from the growing graft. However, the systematical study of the gradual enlargement of the spleen urged to finally admit a growth of the embryonic spleen tissue in loco. This local growth, at least in the beginning, seemed to be caused by an intense uniform stimulation of the mesenchymal cells and of the lymphoid hemocytoblasts and their further differentiation. After the hypertrophic character of the enlargement of the spleen had been established, it was proposed on the basis of this fact to test the validity of the monogenetic conception of the blood origin. If true, the monophyletic interpretation implied analogous changes in other hematopoietic organs as seen in the spleen. The chief character of the changes observed in an embryo, after grafting of an adult spleen, consists indeed in a stimulation of the whole hematopoietic tissue. The hematopoiesis, stmiulated experimentally follows strictly the fundamental principles, estabHshed for birds and reptiles during embryonic and adult life. The hematopoiesis develops however in a peculiar way; first in that groups of cells, which normally would slumber indefinitely, become involved in the process; secondly, that an outburst of hematopoiesis is incited at a time when the normal hematopoiesis is conveyed in definite channels; and finally, that various directions of differentiation are displayed by hemocytoblasts in places where they are usually absent. The study of the changes in the spleen after grafting will form a basis for a comparative study of the changes, incited by the same intervention in other hematopoietic organs.

Since the stimulation leads to different changes according to the stage at which it has been applied, the results of the stimulation in early and later stages will be described separately. As mentioned, grafts of hematopoietic tissue on the chick allantois take easily, if grafted from the end of the 6th day of incubation and on. The grafts, made before the establishment of arterial vascularization in the spleen, incite in the spleen anlage uniform proliferation, which may considerably vary in intensity. The changes are different if the stimulation has been applied after the arterial vascularization has taken place.


a. Changes after stimulation in early stages

. Transformation of the mesenchymal spleen anlage into a uniform granulohastic tissue. The study of the normal spleen development has shown that the spleen anlage at the stage of 6 to 8 days consists of a more or less dense mesenchymal tissue in the form of syncytium. The oblong nuclei, characterized by the presence of well pronounced nucleoli and scant chromatic particles lie closely together. At the periphery of the organ the nuclei are more scattered and the tissue looser. The limits of the cells are here also undefinable, for the cells all are united together by numerous short processes. In the peripheral layers of the organ sinuses are already developed, their connection with the veinous circulation is effected and the stimulating substances find easy access into the organ.

The first effect of the stimulation is exhibited by an intense proliferation of the mesenchymal cells and numerous mitoses. The sinuses spread swiftly over the whole organ and form a net with large meshes. The protoplasm of the mesenchymal syncytium loses soon its uniform structure and becomes less dense; numerous vacuoles appear. At the same time many of the cells become isolated and lose their connection with the plasmodial cell mass. These cells appear intensely basophylic and are very ameboid in their character.^

An intense development of lymphoid hemocytoblasts is seen on figure 6. The differentiation of lymphoid hemocytoblasts can assume such proportions, that the greatest part of the mesenchymal anlage may be converted into free ameboid cells and merely a few mesenchymal cells may remain between them (fig. 20). No regular development of sinuses can take place in such cases. The lymphoid hemocytoblasts multiply intensely, many of them show a beginning differentiation into granulocytoblasts and acidophylic small granules appear in their

^ The changes which occur immediately after stimulation at early stages of embryo development merely accelerate and intensify the normal histogenetic processes in the spleen. Therefore illustrations given on plate 3, figures 5 and 6, refer at the same time to normal development as well as to development after stimulation.



cytoplasm. Numerous spleens of embryos, which were grafted in early stages were transformed 4 to 5 days after grafting into a tissue, similar to that represented on the figure 20.

The ameboid cells appear here pressed closely together, are often of polygonal form and fill up all the interstices between the few sinuses. This uniform wdde-spread conversion of the spleen anlage into a hemogranuloblastic tissue offers some points of general interest. These changes show first, that the action of the stimulus applied is intense and imperative. Products of metabolism appear in the blood current from the growing graft cells. These substances incite an intense proliferation of the mesenchymal cells in the spleen anlage and swiftly transform the syncj^tial Plasmodium of the anlage into an accumulation of ameboid cells, which proliferate and differentiate further. The results may remind one of the experiments of Rous,'* in which connective tissue cells became spherical under the action of trypsin.

As mentioned above the transformation of the mesenchymal cells into lymphoid hemocytoblasts in the spleen anlage may be universal, if stimulation is applied in early stages and merely a few mesenchymal cells may remain undifferentiated, while in normal development numerous mesenchymal cells persist and give in course of time the reticulum tissue. MolUer (28, '11) describes this tissue as a cellular syncytium with a fibrous net adjusted to the cells. The changes in the experiments reported are so intense (fig. 20), that it may be right to assume that numerous cells, which under normal conditions would have passed into the reticulum tissue, now become hemocytoblasts and differentiate further into granulocytoblasts. This result can be taken as a corroborative proof for the polyvalency of the mesenchymal cells, which may either differentiate into . granular leucocytes or become a constituent part of the reticular tissue. The general granuloblastic differentiation of the mesenchymal spleen anlage is observed in embryos to which the stimulation was applied before a development of vascularization has taken place.

^ Personal communication.


Development of intense erythropoiesis after stimulation. — The stimulation of the lymphoid hemocytoblasts to intense proliferation may influence the development of the vessels and of their contents in the mesenchymal spleen anlage. As above stated, the stimulation in early stages leads often to defective development of veinous sinuses and large parts of the spleen anlage are transformed into granuloblastic tissue. If stimulation is applied a little later, at the ninth day, when a number of sinuses are already formed, others still appear, a development of intense erythropoiesis may be observed in the spleen anlage. The normal spleen tissue at this stage consists of a mesenchymal tissue, in which numerous hemocytoblasts continue to develop. The stimulation of the lymphoid hemocytoblasts and the simultaneous opening of splits in the mesenchymal tissue leads to the appearance of numerous hemocytoblasts within the vessels. Larger groups of lymphoid hemocytoblasts may occupy the lumina of the sinuses and here undergo an erythroblastic differentiation. Figure 15 shows a similar split developed locally in the spleen mesenchyme. It is of irregular form and surrounded by mesenchymal cells. These cells enter as a constituent part into the general spleen tissue and send some of their processes within the lumen of the sinus. A development of numerous lymphoid hemocytoblasts is observed in this tissue (fig. 15 E.L.Hhl.) and large groups of l^^mphoid hemocytoblasts are discharged in the lumen of the sinuses (fig. 15, I.L.HbL). Outside the vessels the lymphoid hemocytoblasts differentiate into granulocytoblasts (fig. 15, Grhl.), as elsewhere, within the ^'essels they develop into erythroblasts (fig. 20, Erbl.) and erythrocytes {Ere). The process of the normal differentiation of a lymphoid hemocytoblast into an erythrocyte was studied in birds by Danchakoff (9) and corresponds to what is seen under the condition of an experimental stimulation. Therefore I refer in this respect to my previous papers on the development of different hematopoietic organs in birds and reptiles.

Figure 16 shows the spleen tissue in later stages (four days after stimulation of an eight day embryo) . The veinous sinuses appear surrounded by flattened mesenchymal cells. The inner


surface of the sinuses appears even and regular and is covered by cells which resemble endothelial cells. The vessels still contain a large number of young undifferentiated blood cells which continue to proliferate and to differentiate. The tissue between the vessels begins to assume the character of a reticular tissue, in the meshes of which numerous free ameboid cells are lying. They chieflj^ consist of lymphoid hemocytoblasts, granulocytoblasts, and granulocytes (fig. 16, E.L.Hbl., 6rhl, Grc) together with cells at intermediate stages of differentiation. However a few macrophages may occasionally be seen.

The intense development of erythropoiesis and the activation of granulopoiesis convert the spleen at this stage into a true myeloid organ. As above stated, an occasional differentiation of lymphoid hemocytoblasts into erythroblasts may be observed during normal development of the spleen. Under stimulation large groups of lymphoid hemocytoblasts fall into the developing sinuses. At this time the vascularization in the spleen appear as a net of large veinous capillaries annexed to the portal system and the blood current must be here very slow. Situated in the sinus lumen the lymphoid hemocytoblast is not conveyed away by the blood current, but it remains in the sinus, proliferates and differentiates into a red blood cell. The conditions of the development of the erythropoiesis in the spleen correspond to those known in the 3'"olk sac annexes and in the bone-marrow. A development of large veinous capillaries with a slow blood current and a close connection between erythro- and granulopoiesis is seen in all these regions.

However, the erythropoiesis incited by the experimental stimulation does not persist in the spleen permanently. When the arterious vascularization develops and the connection with the sinuses is effected, the blood current becomes swifter, the younger cells are withdrawn and gradually replaced by differentiated erythrocytes. In other cases in which the arterial vascularization develops defectively and no regular food supply is established a lack of nutritive material finally may determine the suspense of erythropoiesis. Large macrophages develop in the sinuses and an intense phagocytosis of erythrocytes may be observed (figs. 17, 18, 19).


The intense development of erythropoiesis at the expense of lymphoid hemocytoblasts, which develop locally is another striking evidence for the truth of the monogenetic conception of the blood development and consequently for the polyvalency of the mesenchymal cells in the spleen anlage. The same mesenchymal cells, which normally would remain between the vessels and either become a part of the reticular tissue or differentiate into granular leucocytes now differentiate into red blood cells after they are discharged into the vessels.

Deficiencies in the development of vascularization and their ej — The deficiencies in the vascularization of the spleen anlage occur as results of the changes in the spleen incited by stimulation of the stem cells. They can affect either the development of the veinous net or the ingrowth and the distribution of arteries.

The stimulation applied at the 7 to 8 day of incubation may convert the whole spleen anlage or considerab e parts of it into granuloblastic tissue. The sinuses develop thereby defectively and considerable accumulations of granuloblastic tissue are often provided merely with scant and narrow capillaries. The scarce development of veinous sinuses is the natural sequence of the excessive differentiation of granuloblastic tissue. The study of the normal spleen development has shown, that the sinuses develop locally as splits amidst the mesenchymal tissue. Since the greatest part of mesenchyme may be transformed into accumulations of free ameboid cells (fig. 20), the net of sinuses cannot locally develop between the free cells.

Defects in the development of veinous sinuses become themselves the source of interesting alterations in the spleen tissue. The normal histogenesis leads to a gradual differentiation of granular leucocytes at the expense of granuiocytoblasts. The granulocytoblasts, though ameboid, do not migrate normally in the vessels which evidently do not contain adequate chemiotactic substances. The granular leucocytes, however, penetrate easily into the veinous sinuses through thin walls (fig, 11 > Z) and are drifted by the blood current away. Though the differentiation of granular leucocytes is in a normal embryonic spleen continuous,


they never accumulate in the tissue. The conditions develop quite differently after stimulation, and lead to an excessive granulocytspoiesis (figs. 20 and 21). In this case two factors work together in one direction — fii'st: the granular leucocytes are formed in excessive numbers and secondly vessels develop defectively. Considerable accumulations of granular leucocytes remain therefore in the spleen tissue. They may densely infiltrate the mesenchyme, if there is mesenchyme tissue left (fig. 21).

On the other hand, they may form enormous agglomerations in the form of spherical masses of semi liquid tissue and finally perish (fig. 22, Grc.'"). Centers of necrotic tissue appear in the spleen as a result of the excessive production and stagnation of granular leucocytes. The development of large accumulations of granular leucocytes are interesting in so far as it seems to indicate that in the particular case cells cannot stop in their development. The reactions displayed by these living cells necessarilj' lead the cell to the last stage of its differentiation.

The universal and imperative conversion of the spleen mesenchyme into granuloblastic tissue also offers an example of tissue reaction, which being a response to the stimulation, seems to lack the character of purposefulness. The stimulation breaks up the reciprocal normal proportions of the development processes in the spleen. The exclusive development of granulopoiesis in the spleen-anlage leads to formation of considerable centers of completely differentiated cells which finally succumb in large masses. The embryos, in which such wide spread changes are observed, do not hatch, and usually die after 16 to 19 days of incubation.

Around the necrotic centers, if they are not too considerable in size and numbers, a characteristic reaction of mesenchymal tissue may be observed. The mesenchj^mal cells proliferate and form plasmodial masses in the form of giant cells around the necrotic center (fig. 22, Gtx). The specific appearance of the nuclei accumulations may suggest here also the idea of occurrence of amitosis (fig. 22, Y). These giant cells are perfectly similar to those encountered around the foreign bodies. Though their


appearance seems to be purposeful, I do not feel right to take them for such. It seems more correct to attribute their appearance to a reaction similar to that which characterizes the development of macrophages — reaction to particulate matter, Evans (15). The considerable hypertrophy of their cytoplasm is brought by digestion of phagocytozed material. Smaller necrotic centers can undergo a complete resorption by giant cells. The formation of giant cells around necrotic centers can serve as an example of a new differentiation potency assigned to mesenchymal embryonic cells.

Besides deficiencies in the development of veinous vascularization, irregularities in the development of arterious vascularization may also be observed. A partial lack of development of arterious ramifications in districts of the spleen is a frequent occurrence under the conditions of the experiments. A partial deficiency in the veinous vascularization usually coincides and this leads to a nearly complete lack of vascularization in parts of the spleen. In such regions mesenchymal cells are still capable of proliferation, but they soon develop very differently than they do normally, namely^ they become fibroblasts. Large regions of the spleen can be transformed into typical connective tissue (fig. 23). The elongated cell bodies may appear pressed closely together, in other instances they are separated by accumulations of collagenous fibers. A few mitoses are usually observed in these cells. Sometimes the presence of a few lymphoid hemocytoblasts or granular leucocytes may indicate a more intense hematopoietic differentiation which previously has taken place in such regions.

The development of fibrous tissue in the embryonic spleen completes a .whole range of transformations to which the young mesenchymal cells are capable. The leucocyte, the erythrocyte and perhaps the fibroblast and the macrophage are under normal conditions final non interchangeable diffei-entiation stages; the endothelial, the giant and reticulum cells are different morphological structures, which a mesenchymal cell may assume. All these cells derive from one stemcell, and its differentiation depends upon the various conditions to which the polyvalent cell is submitted.


b. Changes obtained by stimulation in the stage with defiriite spleen vascularization

Studying the myeloid metaplasis. Hertz (20) found that besides an intense development of granuloblastic tissue in the pulpa, well pronounced changes in the follicles appeared also. The tissue of the whole follicle might have undergone a differentiation into large lymphocytes (lymphoid hemocy toblasts) . The changes observed in the spleen after stimulation in later stages (at 14 to 15 days of incubation) in which the structural peculiarities of the organ are developed, correspond closely to those described by Hertz. The large lymphocytes (lymphoid hemocytoblasts) develop, according to Hertz, in the follicles partly at the expense of the reticulum cells, partly at the expense of small lymphocytes. The development of lymphoid hemocytoblasts at the expense of the mesenchymal reticulum is most evident in the embryonic spleen after stimulation. However, the present experiments offer no evidence that they develop at the expense of small lymphocytes, because they appear at a time when the small lymphocytes are either still very scant or have not yet developed at all.

Figure 24 represents the tissue of a follicle 3 days after stimulation, applied to a 12 days embryo. The largest part of the cells consists of lymphoid hemocytoblasts, which multiply intensely in a hetero- and homoplastic way. The artery walls themselves are usually infiltrated by large basophylic cells, though normally they are formed at this time by a loose tissue, in which free cells are absent. The splitting off of lymphoid hemocytoblasts by the mesenchymal reticulum in such follicles is easily discernible. Though this splitting leads to the development of numerous hemocytoblasts, it however never attains the intensity observed in the pulpa-hke spleen (fig. 20) after stimulation at early stages. Between the ameboid cells a distinct net of mesenchymal cells is apparent and they proliferate and continue to split off hemocytoblasts. Endothelial cells of the arteries and their smaller branches seem to be exempt from the stimulating action. Whether the lack of a reaction in the endothelial cells is caused by the final specialization of these cells, or whether the endothelial cells are merely


slow ill tlieir response, cannot be definitely determined on the basis of the available experimental material. A similar intense development of lymphoid hemocytoblasts at the expense of reticulmn cells is observed in the pulpa. Thus the stimulation applied at a stage with develo])ed spleen vascularization incites equal reactions through the whole organ. Both in the pulpa and in the follicles this reaction is manifested by an intense sphtting off of lymphoid hemocytoblasts and their intense proliferation.

A study of further development of such spleens shows, however, that the differentiation of the lymphoid hemocytoblasts develops differently according to their localization in the pulpa or in the foUicle. The study of the normal histogenesis has demonstrated that lymphoid hemocytoblasts, situated in the cavernous tissue of the pulpa, differentiate finally into granular leucocytes; in the reticular tissue of the follicle they develop into small lymphocytes. The same strong dependence of differentiation of lymphoid hemocytoblasts upon the environmental structural conditions is observed in a spleen after stimulation. The stimulation stirs up the less differentiated cells, which are the mesenchymal cells and the lymphoid hemocytoblasts. The further differentiation is effected in compliance with the conditions met and granular leucocytes are differentiated in the pulpa, and small lymphocytes in the follicles.^

  • It is difficult to harmonize with the results of my present paper, the data

given by Dr. Murphy in his recent note regarding the effect of adult spleen grafts on the organism of the hostembryo (Journ. of Exp. Med. July, 1916) in which he states that, "while the spleen of a normal embryo of this age (18 days) presents only a beginning differentiation of cells, after grafting this process is well advanced and numerous cells of both the granular and non-granular type are found."

.\s seen in figures 7, 8, 9, 10, 12 and 13 of the present paper taken at the 11th, 13th and 15th days of incubation a normal spleen presents a highly advanced differentiation of granular cells from the 11th day and a good start of development of small lymphocytes from nearly the 15th day of incubation.

The changes in the host spleen after grafts at the 17th and 18th day of incul)ation, 10-11 days after grafting (the only stages to which Dr. Murphy refers in the above quoted note) present the ultimate results of intense modifications, which are very different from the advanced stage of differentiation mentioned by Dr. Murphy.

A remarkable stimulation of granulopoiesis can be seen however in early stages.— at the 2nd, 3rd, and 4th day after grafting. A stimulation of development of small lyin])hocvtes has not been observed as yet.


There were repeatedly described cases of myeloid metaplasis, in which the chief character was represented by a development of granuloblastic tissue and a consequent suppressing of lymphoid tissue. For the true understanding of similar conditions one may remember that granulocytoblasts only might have undergone a stimulation. Granulocytoblasts are specifically differentiated cells, the proliferation faculty of which may be aroused by specific agents, which could leave inert the other offspring of the common stem cells and the stem cells themselves.

The general proliferative reaction of the younger undifferentiated cells to the stimulation observed in the present experiments, no matter where these stem cells are localized^ — whether in the pulpa or in the follicle — point again to a similarity of these cells in their simultaneous reaction to the same factor. It seems, therefore, that to morphological and histogenetic data, new data of uniform biological reaction may be added as evidence for the uniformity of the hemocytoblastic cell group. This biological reaction consists in an intense heteroplastic and homoplastic multiplication as response to the stimulus. Isomorphism, isogenesis and isodynamism under equal conditions, evidently associate in the existence of the lymphoid hemocytoblasts.


On the basis of observations and experiments described in the present paper some data may be won concerning (1) the histogenesis of the blood cells in the spleen, (2) the cell differentiation, and (3) the general meaning of the myeloid metaplasis.

The data concerning the histogenesis of the spleen cells were recapitulated at the end of every section, therefore, I may here merely outline the general conclusions.

The chief results of the study of normal spleen development is the statement of a regular and unalterable relation between differentiation of certain kinds of blood cells and structural environmental conditions.

These results have been conf rmed by the experimental part of the work. Moreover it has been shown experimentally that


tlie development of mesenchyme and lymjilioid liemocytoblasts can be intensely stimulated and that the respective boundaries, in which the differentiation of certain cell groups usually takes place, can be shifted (displaced), in other words, that the prospective potency (after Driesch's terminology) of the blood stemcells is greater than their prospective value.

The fact that under certain conditions nearly the whole amount of the mesenchymal cells of the spleen anlage may undergo a granuloblastic differentiation; that under other conditions they show a fibroblastic, or an erythroblastic differentiation, and so forth, is the natural sequence of the polyvalency of the m.esenchymal cells in the spleen anlage. The early process of segregation must have led in the spleen anlage to a production of one group of numerous homogeneous stemcells, which under different conditions split off varioush' differentiated cells.

Data concerning the conception of cell differentiation

The histogenetical studies of the spleen under normal conditions and after stimulation have shown that a mesenchymal cell, which normally would contribute to the formation of reticulum tissue, can develop into a hemocytoblast or into a fibroblast or a giant cell, that the lymphoid hemocytoblasts can develop into a granular leucocyte, or into an erythrocyte or a small lymphocyte. The study of hematopoiesis in birds and reptiles shows very definite conditions for each of the lines of differentiation. Though Haff (18) has lately described the presence of extravascular erythropoiesis in the liver of hens, yet I was not able to confirm his data. My personal observations concerning the existence of small centers of erythropoiesis in the connective tissue of the hen do not seem to contradict the general conditions. These centers, scattered irregularly and finally phagocytosed may be interpreted as locally developed blood and vessel anlages, of which the connection with the general circulation has not been fully effectuated.

It is known, however, that both erythropoiesis and granulopoiesis develop in mammals extravascularly and, according to Maximow (25) ('11) — "zweineben einander liegende ganz gleiche



ft. Jtn

,/ Jj jjI^).

z. ^


Vera Danchakoff

Text fig. 1. Scheme of cell differentiation and multiplication by equal division gives as result a range of differentiated cells.

\j %^ m, w.,,

-' /



./t/.. ^^^^^t^i^cyi

Vera Dancliakoff

Text tig. 1. Sclieme of cell differentiation and multiplication by unequal division gives as result a range of differentiated cells, a number of stem cells and intermediate stages of differentiation.


Lymphocyten, die sich ja sicherlich auch in gleichen aussern Exist enzbedingungen befinden, doch zu verschiedenen Endprodukten entwickeln." Maximow rightly points out, that the problem of the differentiation of blood cells forms merely a partial question of the general problem of differentiation of an egg in a multicellular organism. The observation that cells begin their granuloblastic differentiation soon after completion of mitosis, led Maximow to connect differentiation with mitosis. The differentiation factor is conceived by Maximow as "eine tiefe Gleichgewichtsstorung" which occurs during cell division is not reversible, and concerns both daughter cells. It leads to specific qualitative changes, — to the development in the cytoplasm of hemoglobine or granules — Warum aus einem sich teilenden Lymphocyt in dem einen Fall ein Paar junger Myelozyten, in einem anderen ein Paar junger Erythroblastenu.s.w.hervorgeht, hangt wahrscheinlich vom Zufall ab. " It is, however, not determined how the chance can lead to definite and well pronounced differences in the development of two identical cells which seem to be submitted to equal surrounding conditions. Maximow' s hypothesis attributes the differentiation to 'Gleichgewichtsstorung,' which depends upon chance. It seems to me that the factor of differentiation which so evidently appears in birds and reptiles, and consists here in definite structural conditions, cannot be essentially different in maimnals. However, these conditions are more difficult to trace in mammals.

The proliferation, of the mesenchyme and lymphoid hemocytoblasts — and perhaps the development of lymphoid hemocytoblasts at the expense of mesenchymal cells — ^seems to depend upon stimulation by enzyme-like substances, which appear in the organism of the embryo from the growing graft cells. The circulating substances call forth an enormous production of lymphoid hemocytoblasts through the whole spleen tissue. In early stages, when the spleen tissue is homogenous and the conditions correspond to those which are required for granulopoiesis, only granular leucocytes are developed, and the differentiation of small lymphocytes does not appear until structural conditions develop in the spleen under which normally


small lymphocytes are differentiated. According to new environmental conditions the mesenchymal cells may develop into typical connective tissue. Again giant cells appear as a reaction of the mesenchymal cells around the necrotic centers. Thus the study of the spleen development in normal conditions and after stimulation adds strong evidence for the conception that at least one of the factors for the differentiation of the polyvalent stem cells consists in the physicochemical conditions to which the cell is submitted.

If the environmental conditions which determine the various differentiation of the stem cells are easily traced, it is much more difficult to understand how differentiation of the stem cells takes place simultaneously with their uninterrupted multiplication. The process of differentiation affects both the cytoplasm and the nucleus. Specific substances are developed in the cytoplasm. The nucleus during differentiation process loses gradually its typical, nucleolus and accumulates chromatin, which permanently remains in the form of intensely basophylic particles. Maximow's recourse to the Gleichgewichtsstorung" during mitosis cannot explain the persistence of the stock of the young stem cells. May the persistence of the young stem cells be explained perhaps by a higher rate of cell multiplication in comparison with their differentiation? I do not think so. If the differentiation is a result of the influence of certain conditions upon the cell, whatever rate of cell proliferation may be admitted, certain kinds of cells under definite conditions will all differentiate simultaneously. The simultaneous and permanent differentiation and multiplication of the lymphoid hemocytoblasts cannot be explained by a high rate of cell proliferation .

A group of hemocytoblasts (let us say a group of similar A cells) develops in the loose connective tissue. Some of these cells differentiate into B cells, or granulocytoblasts; others continue to multiply as such. If the environmental conditions, as they seem to appear, are similar for all these cells, how is the difference in their behavio.ur to be explained? Is a difference in the constitution of different cells involved and does the group of


differentiating cells, A, consist of a number of Aa, and oi Ab cells? The Aa cells may be supposed to be excluded from the differentiating influence of the environmental conditions and multiply as such. The Ab cells may possess the faculty of developing acidophylic granules and become granulocytoblasts. What will then happen? The Aa cell will divide and give two Aa's, the Ab cell will differentiate into a B cell. At what time, then, does the differentiation of an A a cell into an ^6 cell take place? If between two mitosis, then it is to be expected that under definite conditions at a given time all the Aa cells will differentiate into Ab cells and further into B and C cells (text fig. 1.) Yet the stock of A cells does not show any signs of exhaustion and the hemocytoblasts preserve their existence as such.

The simultaneous differentiation and proliferation of young cells could hardly be explained otherwise than by a specific process of differentiation during mitosis (text fig. 2.) The division of a cell A must lead to the development of a cell A and a cell A^. The cell A will continue to give rise to cells A and A^ and will truly become the inexhaustible source of the young undifferentiated cells. The cell A^ will undergo further differentiation and will develop finally into a B cell, or a granulocytoblast, which again will divide into B and BK The cell B^ will differentiate into a C cell, or a granular leucocyte. The differentiation of the hemocytoblasts into granulocytes outside the vessels as well as their development into erythroblasts and erythrocytes within the vessels and their simultaneous inexhaustible proliferation could hardly be explained on other grounds.

I have not been able yet to trace any difference between two daughter cells. However, numerous examples of similar unequal cell division may be found in the life history of other cells. Boveri represents the differentiation of germ and somatic cells in Ascaris as due to cleavages, which result in formation of cells, of which a part conserves the whole chromatine and another loses a considerable amount of it. Again the differentiation of various cell ranges with persistence of the stem cells in Clepsines depends upon unequal division of the stem cells.*' Conklin

« This example was kindly given to me by Prof. H. H. Donaldson.


sees in the cytoplasm division itself a differentiation factor, which accompUshes the segregation of different substances in the cytoplasm. Lately interesting observations were made in the laboratory of Prof. C. McClung by Dr. Wenrich and Miss Carothers, on formation of heteromorphic chromosomes in the spermatogenesis of grasshoppers. The admission of similar unequal chromosome-division in somatic cells may explain the gradual development of different mitosis figures characteristic of different tissues. The small sizes of the somatic cells do not allow to gain direct data on the inequality of the daughter cells. However, the examples cited above sufficiently explain the possibility of simultaneous persistence of young stem cells and their further differentiation.

In connection with the problem of cell differentiation, the existence of a large number of amitotic cell divisions, may be mentioned. Lately Patterson has described in the Keimblattern of the pigeon, the presence of numerous amitoses. In regions of intense proliferation the cells seemed to undergo a full amitotical division, and give rise to apparently normal daughter cells, which could themselves multiply mitotically. Maximow (25) ('08) found similar conditions in certain regions of embryos in mammals. In early stages of spleen development in chicks, when the nuclei proliferate intensely in the mesenchymal spleen anlage, numerous pictures of amitotical nucleas division may be encountered. Figure 14 illustrates four cells in a stage of amitotic nuclei division. As a result of the division of the nucleus two daughter nuclei arise, which can have both the same dimensions, or differ considerably in size. Both daughter nuclei receive always a part of the nucleolar substance. In most cases the nuclei divisions are not followed by cell divisions, for the very fact that their localization in the syncytium mass of the mesenchyme does not allow a division of the cytoplasm. However, sometimes free 'cells may be observed undergoing amitotic nuclei division (fig. 13, 14 d). The further destiny of such nuclei cannot be followed directly. The frequence of occurrence of amitotic nuclei division must, however, lead in the spleen mesenchyme to a production of a larger number of nuclei, derived through amitosis. These nuclei contribute in a great part to the growth


of the apparently homogeneous mesenchymal anlage, at the expense of which many different ceils are developed. I cannot enter now into a detailed consideration of this problem. However, the question whether the amitosis may not be looked upon as one of the factors in the differentiation of the living substance, can find its justification.

Data concerning the general meaning of the myeloid metaplasis

The results obtained in the embryonic spleen at different stages of its development have a close bearing upon the myeloid metaplasis, well known to the pathologists. What is the general meaning of these changes, and what are their relations to the stimulus applied and to other reactions displayed at the same time by the organism? The specific conditions under which I had to work last winter did not allow me so far to obtain definite data in this respect. However, the study of the experiments made may give a few suggestions concerning the problem.

There is no doubt the intervention applied introduces in the organism heterogeneous substances. The connection between the introduction of these substances and the changes described above may be conceived in two different ways. Either these substances have general stimulating action on the mesenchyme and on the blood stem cells (as for example the thyroid substance in the experiments of Gudernatsch^ on the development of tad-pole limbs) ; or the action of these substances may be similar to that of specific antigenes, which introduced in the organism incite the production of antibodies. In the latter case the proliferative reaction exhibited by the blood stem cells may be looked upon as a material basis for the phenomena of immunity. The proliferation in this case would have been brought about by the interaction between specific antigenes and their cell receptors. Only results of new series of experiments will decide which of these two conceptions has to be accepted.

The analogy between the myeloid metaplasis and the results of the experiments described is obvious. It is important to

^ Gudernatsch, F. Feeding Experiments on Tadpoles. II, A further contribution to the knowledge of organs with internal secretion, Am. Jour. Anat., 1914, vol. 15.


notice that the myeloid metaplasis is produced by different causes. The toxines of various bacteria, the specific products of metaboHsm of mahgnant tumors, finally, inorganic chronic intoxications may incite an extensive myeloid metaplasis. It is difficult to conceive in such qualitatively different agents a specific stimulating influence on the stem cells. The response to the action of these factors is specific in so far as it is exhibited by a certain kind of tissue (even not of cells). The stimulus itself may largely vary. The cell, being understood as a complex group of receptors, may offer adequate receptors to different stimuli or antigenes. A similar example of stimulation to proliferation by different agents may be found in the phenomena of fertilization. The specific or usual stimulus in the form of the spermatozoon may be replaced by other chemical stimuli, which may find adequate receptors in the egg cell and incite therefore molecular changes, which are followed by proliferation.

If more than occasional coincidence is to be seen in the regular connection of the appearance of different antibodies and the myeloid metaplasis after infections, the specific antibodies may consist of substances derived from the proliferation and differentiation activities of the hematopoietic tissue. If so, a mere stimulation of the hematopoietic tissue would suffice for strengthening or developing immunity; the production of a large amount of antibodies would follow as a result of stimulation plus specific action of the antigene. Otherwise how could be explained the development of immunity against heteroplastic grafting by introduction in the organism of an emulsion of various tissue (Da Fano)? This intervention, similarly to the experiments described, must have also stimulated the hematopoietic tissue.

It is too early now to attempt to draw more definite conclusions concerning the specific functions of the hematopoietic tissue. Confronted with the simultaneous development of widespread changes in the mesenchyme, which occur after the appearance in the organism of antigen-like substances and usually followed by a production of specific antibodies, one may find it natural to think of the mesenchyme and its differentiation products as of an organ in close relation to the production of immune bodies.


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(22) Klein, St. 1914 Die Myelogonie. Berlin, Julius Springer.

(23) Laguesse, E. 1890 Recherches sur le developpement de la rate chez les

poissons. Journ. de I'Anat. et de la Physiologic, 26 Annee.

(24) Lobenhoffer, W. 1908 Uber extravaskulare Erythropoese in der Leber

unter path. u. normalen Verba Itnissen. Ziegler's Beitrage, Bd. 43.


(25) Maximow, a. 1907 UIxm- die Kutwickluiig der Blut- uiid Bitidergewobs zellcn bcim Saiigetiercmbryo. Folia Haeinat. Bd. 4, N. 5.

1908 Amitosis.-Anatom. Anz. Bd. 33.

1909 Untersuchungen iiber Blut und Bindegewebe. I- Die friiheste Entwickluiigsstadien etc. Arch, fiir Mikr. Anat. Bd. 73.

1911 Untersuchiingen i'lber Blut und Bindegewebe. Ill- Die embr. Histogenese des Knochcnmarkes dor Saugetiere. Arch. f. Mikr. An. Bd. 76.

(26) Meyer itnd Heinecke, A. 1907 Uber Blutl)ildung bei schweren Aniimien

und Leukamien. Deut. Arch, fur Klin. Med. Bd. 88.

(27) Meyer, E. 1908 Weitere Untersuchungen fiber extra-uterine Blut bildung. Mi'inch. Med. Wochenschr. N. 22.

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der Saugetiere. Arch, fur Mikr. Anat. Bd. 74.

1911 tJber den Ban der Kapillaren der Milzvenen. Arch. f. Mik. An. Bd. 76.

(29) Murphy, Jas. B. 1913 Transplantability of tissues to the embryo of

foreign species Jour, of Exp. Med. vol. 17.

(30) Murphy, Jas. B., and Rous, P. 1912 The behaviour of chicken sarcoma

implanted in the developing Embryo. -Journ. of Exp. Med. vol. 15.

(31) Nageli, O. 1906 Beitriige zur Embriologie der blutbildendenden Organe.

Verh. Kong Inn. Med.

1912 Blutkrankheiten und Blutdiagnostik. 2 Auflage. Leipzig.

1913 Blut. Aschoff's Pathologische Anatomic. Bd. 2.

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1908 liber lymphoide basophile Vorstufen der Erythroljlasten. Folia Haemat. Bd. 5.

1909 Pathologie und Therapie der Leukamien. Prolegomena. Fol. Ham. Bd. 7.

1909 Zur vorstehenden Mitteilung Dominici's Fol. Haem. Bd. 8. 1911 Kurze technologische Zusammenstellung der Farbungsforschrift mit Panchrom. Folia Haemat. Bd. 11. 1913 Prolegomena. Folia Haematol. Bd. 14.

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1911 Uber die akute myeloische Leukamie. Wien. Klin. Wochenschr,

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(42) TuRCK, W. 1912 Vorlesungen uber klinische Hamatologie. Wien.

(43) Venzlaff 1911 Uber Genesis und Morphologie der roten Blutkorperchen

der Vogel. Arch, fur Mikr. Anatom., Bd. 77.

(44) Weidenreich, F. 1901 Das Gefasssystem der menschlichen Milz. Arch.

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(46) Werzberg, a. 1911 Neue experim. Beitr-ige zur Frage der myeloiden

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Histogenese der myeloiden Leukamie. Jena.

1908 Uber die Beziehungen zwischen myeloiden L'mwandhmg und

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AH the figures were drawn with the camera lucida at stage level with Zeiss Apochromat 2 mm. oil immersion obj. The compensatory ocular 4 was used for the figure 10, the oc. 6 for the figures 7, 8, 9, 11, 12, 13, 23 and 24, the oc. 8 for the figures 15, 16, 17, 18, 19, 21 and 22 and the oc. 12 for the figures 5, 6, 14 and



Art., artery

Art.c, arterial capillary Erbl., erythroblast Ere, erythrocyte Ere.,'" degenerated erytlirocyte Fbr., fibrous tissue Grbl., granulocytoblast (myelocyte) Grc, granulocyte (granular leucocyte) Grc.,'" degenerated granulocyte Gtc, giant cell

L.Hbl., lymphoid hemocytoblast, with a denomination — mitosis of the corresponding cell

L.Hbl., " lymphoid hemocytoblast in the stage of its isolation from the mesenchymal syncytium

Msc, mesenchymal cells

Ms. St., mesenchymal syncytium

S., sinus

S. L.Hbl., small lymphoid hemocytoblast

S.Lmc, small lymphocyte

I. L.Hbl., intra-vascular lymphoid hemocytoblast

E. L.Hbl., extra-vascular lymphoid hemocytoblast



1 Slight hypertrophy of the spleen, corresponding to the graft, reproduced on figure 3. Eighteenth day of incubation.

2 Enormous hypertrophy of the spleen, corresponding to the graft, reproduced on figure 4. Eighteenth day of incubation.






3 Spleen graft of adult tissue, growing on the surface of the allantois of a chick embryo. The culture gave merel.y a slight growth.

4 The same. Graft intensely growing. The tumor-like graft is well provided with vessels. The culture was made on the 7th day of incubation. The graft is fixed on the 18th dav of incubation, being 9 days old.







5 Part of the spleen, adjacent to the surrounding loose mesenchyme. Large group of hemocytoblasts within the sinus, beginning their differentiation into erythrocytes. Eighth day of incubation.

6 Part of the spleen from the center of the organ. S.AyiL, sinus anlage. Xinth dav of incubation.





S. L Hbl.




-i^. — -,:>^Ms.St

— .^ ^ — ^^. _^







Vera DanchakofiF




7 and 8 Parts of a normal pulpa-like spleen. Eleventh day of incubation. Figure 7 shows newly formed sinuses wdth particularly numerous lymphoid hemocytoblasts inside. Figure 8 shows a part of the spleen with definitely formed veinous capillaries and well developed granulopoiesis between the vessels.

"9 Ingrowth of arterial capillaries into the pulpa-like spleen. Thirteenth day of incubation.










,.r G^bl.

.^. '"

' **#-r-j^'^ -^ ■'■■■




10 Part of a normal spleen, in which the follicle is adjacent to the pulpa.

11 Infiltration of the spleen tissue with granulocytes and immigration of granulocytes into the vessels. Z, immigration of a leucocj^te in the vessel. Eight days after stimulation of an 8 day embryo.






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^ 1. ^




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Vera Danchakoff





12 and 13 Differentiation of small lymphocytes in the follicle anlages. Figure 12 shows the center of a follicle, figure 13 a part of a follicle adjacent to the pulpa. M.Rtc, mesenchymal reticulum; Sl.L.Hbl., group of lymphoid hemocytoblasts forming a kind of syncytium; Y, amitotic division of a nucleus in a reticulum cell; X, intermediate stage between a lymphoid hemocytoblast and a small l>Tiiphocyte. Fifteenth day of incubation.

14 Different stages of amitotical division of the nucleus; a, b, c, in mesenchymal cells, d in a free cell.





,« sJ.Lmc.








^-t%J^'^ ^§s>'\ ^m ^1:^

if Iff" .#^(K>! ■^"^- ' #^V^



^ SLHbl.

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15 Intense erythropoiesis within developing sinuses. Granulopoiesis outside the vessels. Two days after stimulation of an 8 day embryo.

16 The same. Four days after stimulation of an 8 day embryo.

17, IS and 19 Intense phagocytosis of erythrocytes by reticulum and endoth. cells in figure 17, by a polynuclear macrophage in figure 18 and a lymphoid liemocytoblast in figure 19.







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Vera Daiicliakoff



20 Granuloblastic transformation of the spleen aniage o days after stimulation of a 6 day embryo.

21 Leucocytic infiltration of large regions in the spleen 6 days after stimulation of an 8 day embryo.

22 Reaction of the spleen tissue around large necrotic accumulations of granulocytic tissue. Plight days after stimulation of an 8 day embryo.




/ L.Hbl Ore •"-,__


^ '^.. y-^ ,i^^';^-«^^^<'


<^ ■^*^>


^Grc"" ^


I /^- >. .^7 L,Hbl. ^P" ^^- 'W^'i^^



^ J .^


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23 Transformation of the spleen tissue into a fibrous tissue. Ten days after stimulation of a 9 day embryo.

24 Lymphocytoblastic transformation of a follicle 3 days after stimulation of a 15 day embryo.



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Vera DanchakofT

2A 327