Identification and Distribution of Megakaryocyte Colonies in Murine Spleen

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1 International Journal of Cell Cloning 1: (1983) Identification and Distribution of Megakaryocyte Colonies in Murine Spleen Chandra Choudhury, Pamela A llman, Eugene Arnold Departments of Oncology, Medicine, and Pathology; Johns Hopkins University School of Medicine, Baltimore, MD, USA Key Words. Spleen colonies - CFU-S * Differentiation - Megakaryocyte colonies. Acetylcholinesterase.. Microenvironment Abstract. This study is the first report on the utilization of specific cell function to identify splenic megakaryocytic colonies. Stem cell differentiation into megakaryocytes was studied by injecting each irradiated murine syngeneic recipient with 1 X 106 spleen cells. Morphological identification of erythroid, granulocytic, megakaryocytic, and mixed and undifferentiated colonies was done by staining consecutive cryostat sections with hemotoxylin and eosin, benzidine, myeloperoxidase, and acetylcholinesterase. The variation in the distribution of hemopoietic colonies within the spleen was reflected in the different ratio values derived for erythroid, granulocytic, and megakaryocytic colonies at varying depth within the spleen. An increase by 50% of megakaryocyte colonies was seen within the splenic pulp in the midzone region, compared with the surface. This suggests a localized microenvironment conducive for megakaryocytopoiesis. The data emphasizes the importance of identifying colonies of all cell types in histological seo tions of the spleen and evaluating spleen sections at least at two levels, one adjacent to the surface and the other in the midzone area. Introduction Transplantation of normal hemopoietic cells into irradiated murine recipients gives rise to hemopoietic colonies in the spleen [ 11. It has been debated whether the colonies formed are the progeny of unipotent or pluripotent stem cells. An approach to the elucidation of this problem has been the use of histological sections to identify the cellular composition of spleen colonies using techniques which tested for specific cell function AlphaMed Press, Inc /83/$2.00/0

2 Megakaryocyte Colonies in Murine Spleen 390 rather than cell morphology [2]. However, there is no report in the literature on the utilization of specific cell function to identify splenic megakaryocyte colonies. It does appear that spleen colonies formed 10 days after post-irradiation reconstitution are of a permanent nature [3], and are derived from pluripotent stem cells which differentiate into colonies of one predominant cell lineage [4, 51. In this study megakaryocytic colonies were identified by specific cell function 10 days after post-irradiation reconstitution. Megakaryocytes are the only murine hemopoietic cell with acetylcholinesterase (AChE) activity [6,7]. Utilizing AChE activity as a marker, small AChE-positive cells are identified which are not recognized morphologically as megakaryocytes [8-lo]. It also has been reported that following transplantation with splenic stem cells, significantly more splenic megakaryocytes are produced than by transplantation with bone marrow [l 1, 121. We have identified splenic megakaryocytic colonies in cryostat seo tions of spleen by staining for AChE activity. In view of the propensity of splenic stem cells for direction of differentiation along megakaryocytic cell lineage, this method was used to study the composition and distribution of megakaryocytic colonies and their relationship to other hemopoietic colonies, following injection of spleen cell suspensions into irradiated mice. Materials and Methods Animals The mice used in these experiments were 8- to 10-week-old C57B1/6J female mice (Cumberland Farms, Clinton, TN). Following irradiation and injection of hemopoietic cells, the animals were housed in cages covered with air filters, and allowed food and water ad libitum. Control animals, which were matched for age and sex, were irradiated with the test animals. The uninjected controls were housed with the test animals and identified by ear punch marks. Radiation Animals were irradiated with 900 rads, at a dose rate of 136 rads/min in air, in a small animal irradiator (Gamma Cell 40) with dual source cesium irradiator. Cell Suspension Spleen cell suspensions were prepared from syngeneic donors in normal saline (Cutter Laboratories, Berkeley, CA). The viable cell count was determined by trypan blue exclusion in a hemocytometer. One million (106) viable cells, contained in 0.2 ml normal saline, were injected into the lateral tail vein with a tuberculin syringe and a 27-gauge needle. All animals were injected within 2 h following irradiation. The irradiated controls were uninjected.

3 Choudhury / Allman/ Arnold 391 Spleen Colonies and Cryostat Sections Spleen colony formation was assayed 10 days following transplantation. Surface colonies were counted macroscopically. The spleens were then snap frozen in OCT compound (Ames Co., Elkhart, IN) and serial frozen sections, 4 microns (p) in width, were cut on a cryostat (IEC Cryostat, Needham, MA) at -20 C. The spleens were oriented and sectioned lengthwise starting at the surface and proceeding through the width of the spleen. Half of the specimens were sectioned starting from the visceral surface, and the remaining half were sectioned starting from the parietal surface. Four consecutive sections of 4 p in thickness were cut and mounted, the subsequent 20 sections were discarded before the next four sections were mounted. From each series of four sections, one was stained with hemotoxylin and eosin (H and E); and subsequent sections for myeloperoxidase (Histozyme, Sigma Chemical Co., St. Louis, MO), for AChE [ 131, and with benzidine, respectively. Results Surface Colonies Macroscopic surface colonies were 7.8 f 2.7 in six animals. No surface colonies were visible in uninjected controls. Morphologic Identijcation of Hernopoietic Colonies Granulocytic colonies were identified as containing cells with doughnu t-shaped nuclei. The adjacent section, stained for myeloperoxidase, confirmed the presence of myeloid cells in these colonies. Erythroid colonies contained normoblasts, the nuclear chromatin of which stained deep purple with H and E. Benzidine staining of adjacent sections demonstrated benzidine-positive cells associated with these colonies. Mixed colonies contained cells identified as granulocytic and erythroid; occasionally megakaryocytes were seen within these colonies. The multiple cell type composition of these colonies was also seen on the sections stained for specific cytochemistry. However, it was not possible to determine whether the mixed colonies originated as an entity or were formed by infiltration from adjacent colonies. Megakaryocyte colonies were identified on H and E sections as small clusters of recognizable megakaryocytes. AChE stain, however, identified an increased number of megakaryocyte colonies. These large colonies consisted of 80 or more cells. The nidus of a colony could be identified, from which cells spread diffusely into the splenic stroma (Fig. 1). A comparison of the means of AChE-positive colonies with the means of megakaryocyte colonies identified in H and E stained sections gave a

4 Megakaryocyte Colonies in Murine Spleen 392 Fig. 1. Adjacent serial sections of spleen from the midzone area. (A) H & E stained section. (B) Acetylcholinesterase stained section. The arrow indicates the area which has been examined in (D). (C) Few recognizable megakaryocytes are seen on H & E stained section, X 450. (D) Acetylcholinesterase stain identifies most of the cells as AChE-positive megakaryocytes, x 450.

5 Table I. Frequency of colony types in spleen sections at varying levelsa Spleen Non-megakaryocytic colonies: differential and total Megakaryocytic colonies: total section depth Erythroid Granulocytic Mixed Undifferentiated Total H&E AChE POS Non-MEG + AChE-POS MEG f f k f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f 19.4 a Values represent mean f one standard deviation for six animals. Macroscopic surface colonies were 7.8 f 2.7 (n = 6). w W W

6 Megakaryocyte Colonies in Murine Spleen 394 ratio of 2.51 (P < 0.01) at 0-16 p and 1.8:l (P < 0.01) at 500 p. There was a significant increase in the number of megakaryocyte colonies identified by AChE activity (Table I). Specific cytochemistry for myeloperoxidase and benzidine stain did not show a difference between myeloid and erythroid colonies identified by these stains and by the conventional H and E stain. Undifferentiated colonies were classified as those which could not be identified as any of the above colony types, either by H and E stain or specific cytochemistry. Control Animals H and E stain and specific cytochemistry of step serial sections of the control irradiated spleens identified 1 f 1 erythroid colonies (mean It SD) in sections at 16 p to 200 p and 2 f 1 in sections from 300 p to 600 p. No granulocytic or megakaryocytic colonies were seen in any of the controls. Geographic Distribution of Colonies Table I gives the distribution of colonies at varying depth within the spleen. The initial four spleen sections were adjacent to the surface (O- 16 p); subsequent serial sections were at intervals of approximately 100 p. The total number of colonies increased progressively towards the midzone of the spleen, between p and was maximal at 500 p, The number of colonies then decreased with sections proceeding from the midzone towards the opposite surface. No difference was detected in colony distribution when proceeding from either the visceral or the parietal surface to the midzone of the spleen. A histogram (Fig. 2) depicts the relative proportions of the colonies with increasing depth of spleen section. Erythroid colonies formed the major component of total colonies. The increase in the total number of colonies and the erythroid colonies was proportional, which is reflected by the ratio of total colonies to erythroid remaining constant with 2.2:l at the surface and 2.1 : 1 at the midzone (500 p) (Table 11). Granulocytic colonies showed a proportional decrease, while megakaryocytic colonies showed an absolute increase in number, with increasing depth within the spleen. Consequently, the erythroid/granulocytic (E:G) ratio increased from 1.8: 1 (0-16 p) to 3.2: 1 (500 p), erythroid/megakaryocytic (E:M) ratio decreased from 2.8:l (0-16 p) to 1.8:l (500 p), and the granulocytic/megakaryocytic (G:M) ratio reversed from 1.6: 1 (0-16 p) to 0.6: 1 (500 p) (Fig. 2; Table II).

7 ~ Choudhury/Allman/Arnold 395 Fig. 2. The variation in relative proportions of the colonies with increasing depth within the spleen is shown on the histogram. Total colonies represent the sum of non-niegakaryocytic and AChE-positive megakaryocytic colonies. The bars represent one SD. Table II. Ratio of colonies in spleen sections at varying levels Ratio Level of spleen section 16 IL 100 p 200 p 300 p 400 P 500 p T:Ea 2.2: 1 2.0: 1 1.9: 1 1.9: 1 1.9: 1 2.1:1 E:G 1.8: 1 2.2: 1 2.0: 1 4.6: 1 4.0: I 3.2: 1 E: M 2.8: 1 2.5: 1 2.2: 1 1.7: 1 1.6: 1 1.6:l G:M 1.6: 1 1.1:l 0.7: 1 0.4: 1 0.5: 1 0.6: 1 a T = Total, E = Erythroid, M = Megakaryocyte, G = Granulocyte

8 Megakaryocyte Colonies in Murine Spleen 396 Analysis of regression (Fig. 3) was done from the number of colonies for each animal plotted on the ordinate and the level of the respective spleen sections on the abscissa. Erythroid colonies showed a progressive increase in numbers with depth of spleen section. The correlation coefficient (r) ranged from 0.45 to Granulocytic and mixed colonies did not show a correlation with depth of spleen section. Megakaryocytic colonies show a striking increase with depth within the spleen, < 5 microscopic colonies being present near the surface compared with > 15 in the midzone (500 p). Analysis of the values for each individual animal gave a correlation coefficient of > 0.97 for four of the animals. The coefficient of determination (RZ), indicates the goodness of fit (R2 > 0.95). In the remaining two, r = 0.54 and 0.15, respectively, the first value may represent a within-animal variation, in that colonies increased to 300 p, (R2 = 0.8) with a plateau up to 600 p,. Histological examination of the sections from the second animal revealed fibrosis in the midzone area (Fig. 3). This may represent post-traumatic splenic fibrosis. The disruption of splenic architecture is reflected in the lack of correlation of colofiy numbers and depth of spleen section (R2 < 0.1). Since these events may occur in any group of experimental animals, the values have been retained in the data. Discussion Surface colonies are formed in the spleens of irradiated mice following injection with murine hemopoietic cells. Differentiation of colonyforming cells into erythroid, granulocytic, or megakaryocytic cell lineages is recognized by morphological identification in histological sections of the spleen. Megakaryocyte colonies in spleen sections stained with H and E are difficult to identify morphologically. Reports on the morphological appearance of megakaryocyte colonies on H and E sections vary from minute colonies [ 141, to colonies containing anywhere from 8 to 100 megakaryocytes, occurring either adjacent to the splenic capsule or deep within the splenic pulp [15]. The transient nature of spleen colonies formed seven days after transplantation has been reported [3]. In this study, spleen colonies were estimated 10 days following transplantation. Specific staining for AChE activity identified a greater number of megakaryocyte colonies compared with conventional H and E stain. There was a significant increase in the number of megakaryocyte colonies formed with increasing depth within the spleen. Maximal numbers were present in the midzone

9 Choudhury / Allman/ Arnold 397 Fig. 3. Plots for analysis of regression show the number of colonies for each animal plotted on the ordinate. The figures on the abscissa indicate the level of the spleen section, corresponding to 16 p, 100 p, 200 p, 300 p, 400 p, and 500 p respectively. Each symbol identifies an individual animal. (A = animal with spleen fibrosis).

10 Megakaryocyte Colonies in Murine Spleen 398 area, comprising 26.9% of the total colonies in that area. In order to get an accurate morphologic estimate of the distribution and proportion of colonies within the spleen, it is important to identify colonies of all the cell types. The data (Table I) showed that if only non-megakaryocytic colonies, which were readily identified in H and E sections, were considered, they increased in number with depth within the spleen. In this case the erythroid component contributed mainly to the increase, with 54.3% erythroid colonies near the surface (0-16 p) and 64.6% in the midzone area (500 p). However, when the total number of colonies of all cell types was considered (i-e., the sum of non-megakaryocytic and AChE-positive megakaryocyte colonies) again there was an increase in the total number of colonies with depth, with a concomitant increase in the number of erythroid colonies. However, in this instance the proportion of erythroid colonies to total colonies was not changed (Fig. 2; Table I, II). Megakaryocyte colonies, however, showed a fivefold increase with depth within the spleen and the proportion to the total number was doubled (Fig. 2; Table 11) in the midzone compared with the surface adjacent to the splenic capsule. The absolute number of granulocyte colonies, however, remained similar, irrespective of the geographical area; consequently, the proportion of granulocyte colonies relative to the total colonies decreased with depth within the spleen (Fig. 2; Table I, 11). The spleen colony assay is associated with variations between animals due to variable stem cell input, to differences within animals in seeding efficiency (f values), and to possible loss of cells at the injection site. This within-animal variation is reflected in our data in Figure 3. However, these variables do not contribute to variations in the distribution of spleen colonies for each individual animal. The differing distribution of colonies of the various cell types within each spleen was reflected in the different ratio values derived for erythroid, granulocytic, and megakaryocyte colonies, at varying depth within the spleen (Table 11). The data indicated that morphological assessment of spleen colonies should be done at two levels within each spleen, one level of section adjacent to the surface of the spleen and a second level at the midzone area. Serial sections in this study did not show significant difference in the values in sections at p (Table I). Therefore, an approximate midzone level within the spleen would be acceptable. It has been suggested that differentiation of pluripotent stem cells into colonies of one predominant hemopoietic cell lineage reflects the operation of instructive differentiative signals in the splenic microenvironment

11 Choudhury/Allman/ Arnold 399 [16]. The increased number of megakaryocyte colonies deep within the splenic pulp suggests a geographic localization of a microenvironment conducive for stem cell differentiation into megakaryocytic cell lineage. Acknowledgments Dr. Choudhury was supported during part of this study by National Research Award HL07377 from the National Heart, Lung, and Blood, National Institutes of Health. We wish to thank Dr. J. Boitnott for assistance with the statistical evaluation, Wanda Novak for secretarial assistance, and Margaret Condon for graphics. References Till, J.E.; McCulloch, E.A.: A direct measurement of the radiation sensitivity of normal mouse bone marrow cells. Radiat Res 14: ( 1961). Fowler, J.H.; Wu, A.M.; Till, J.E.; McCulloch, E.A.; Siminovitch, L.: The cellular composition of hemopoietic spleen colonies. J Cell Physiol69: (1967). Magli, M.C.; Iscove, N.N.; Odartchenko, N.: Transient nature of early haematopoietic spleen colonies. Nature 295: (1982). Becker, A.J.; McCulloch, E.A.; Till, J.E.: Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells. Nature 197: (1963). Rosendaal, M.; Hodgson, G.S.; Bradley, T.R.: Organization of haemopoietic stem cells: the generation-age hypothesis. Cell Tissue Kinet 12: (1979). Zajicek, J.; Datta, N.: Investigations on the acetylcholinesterase activity of erythrocytes, platelets and plasma in different animal species. Acta Haematol 9: (1953). Zajicek, J.: Studies on the histogenesis of blood platelets and megakaryocytes. Acta Physiol Scand 40: suppl. 138 (1957). Zajicek, J.: Studies on the histogenesis of blood platelets. 1. Histochemistry of acetylcholinesterase activity of megakaryocytes and platelets in different species. Acta Haematoll2: (1954). Jackson, C.W.: Cholinesterase as a possible marker of early cells of the megakaryocyte series. Blood 42: (1973). Long, M.W.; Williams, N.: 'Immature megakaryocytes in the mouse: Morphology and quantitation by acetylcholinesterase staining. Blood 58: (1981). Ebbe, S.; Phalen, E.; Overcash, J.; Howard, D.; Stohlman, F., Jr.: A difference between stem cells from marrow and spleen in initiating splenic megakaryocytopoiesis. Proc SOC Exp Biol Med 137: (1971).

12 Megakaryocyte Colonies in Murine Spleen Bentfield-Barker, M.E.; Schooley, J.C.: Comparison of the effectiveness of bone marrow and spleen stem cells for platelet repopulation in lethally irradiated mice. Exp Hematol9: (198 1). 13 Karnovsky, M.J.; Roots, L.: A direct coloring thiocholine method for cholinesterases. J Histochem Cytochem 12: 219 (1964). 14 Juraskova, V.L.; Tkadlecek, L.; Drasil, V.: Note on the differentiation of colonies of haematopoietic tissue cells in the spleen of irradiated mice. Folia 15 Biol 10: (1964). Lewis, J.P.; Passovoy, M.; Freeman, M.; Trobaugh, F.E., Jr.: The repopulating potential and differentiation capacity of hematopoietic stem cells from the blood and bone marrow of normal mice. J Cell Physiol 71: (1969). 16 Curry, J.L.; Trentin, J.J.: Hemopoietic spleen colony studies. 1. Growth and differentiation. Dev Biol1.5: (1967). Received: March 17,1983; accepted: June 24, 1983 Chandra Choudhury, M.D., Johns Hopkins Oncology Center, B-176,600 North Wolfe Street, Baltimore, MD (USA)