Polyploid Megakaryocytes Research Articles

Growth and Differentiation of the Human Megakaryoblastic Cell Line (ELF-153): A Model for Early Stages of Megakaryocytopoiesis

Abstract ELF-153 is a cell line that has been established from a patient with a poorly differentiated acute myeloid leukemia associated with an acute myelofibrosis. A majority of cells had a blast morphology with the phenotype of a myeloid hematopoietic progenitor, ie, CD34+, CD33+, CD13+, HLA-DR+, but CD38-, and the remaining cells (5% to 10%) expressed platelet restricted proteins such as CD41, CD42, CD36, CD61, and von Willebrand factor; some of them were polyploid (up to 32N) and exhibited demarcation membranes and alpha granules. No erythroid or other lineage-specific markers were detected. Proliferation of ELF-153 cells was highly stimulated by interleukin-3 (IL-3) and granulocyte- macrophage colony-stimulating factor and to a lesser extent by stem cell factor and IL-6. In contrast, the cell line did not respond to erythropoietin, leukemia inhibitory factor, IL-7, IL-11, granulocyte colony-stimulating factor, and basic fibroblast growth factor. ELF-153 cells could be separated by flow cytometry into three discrete cell populations (CD34+/CD61-, CD34+/CD61+, and CD34-/CD61+) with different proliferative and endomitotic properties corresponding to distinct stages of the mega karyocyte (MK) differentiation. This MK differentiation, which involved a minority of ELF-153, could be increased in the presence of 5-azacytidine and phorbol ester, but could not be significantly modified by growth factors. By contrast, cytochalasin B dramatically induced polyploidization without differentiation. It is noteworthy that association of 5-azacytidine to cytochalasin B dramatically induced the production of polyploid MK cells. To understand the molecular mechanisms underlying this MK differentiation, the expression of GATA-1 and GATA-2 was investigated in subpopulations of ELF-153. A high level of GATA-1 and GATA-2 mRNA was only present in the CD61+ cells. Therefore, these two transactivating factors may play an important role in the MK differentiation of ELF-153. We conclude that ELF-153 might be an important tool to investigate the mechanisms by which transcription factors control differentiation of MK progenitors.

Growth and differentiation of the human megakaryoblastic cell line (ELF- 153): a model for early stages of megakaryocytopoiesis

ELF-153 is a cell line that has been established from a patient with a poorly differentiated acute myeloid leukemia associated with an acute myelofibrosis. A majority of cells had a blast morphology with the phenotype of a myeloid hematopoietic progenitor, ie, CD34+, CD33+, CD13+, HLA-DR+, but CD38-, and the remaining cells (5% to 10%) expressed platelet restricted proteins such as CD41, CD42, CD36, CD61, and von Willebrand factor; some of them were polyploid (up to 32N) and exhibited demarcation membranes and alpha granules. No erythroid or other lineage-specific markers were detected. Proliferation of ELF-153 cells was highly stimulated by interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor and to a lesser extent by stem cell factor and IL-6. In contrast, the cell line did not respond to erythropoietin, leukemia inhibitory factor, IL-7, IL-11, granulocyte colony-stimulating factor, and basic fibroblast growth factor. ELF-153 cells could be separated by flow cytometry into three discrete cell populations (CD34+/CD61-, CD34+/CD61+, and CD34-/CD61+) with different proliferative and endomitotic properties corresponding to distinct stages of the mega karyocyte (MK) differentiation. This MK differentiation, which involved a minority of ELF-153, could be increased in the presence of 5-azacytidine and phorbol ester, but could not be significantly modified by growth factors. By contrast, cytochalasin B dramatically induced polyploidization without differentiation. It is noteworthy that association of 5-azacytidine to cytochalasin B dramatically induced the production of polyploid MK cells. To understand the molecular mechanisms underlying this MK differentiation, the expression of GATA-1 and GATA-2 was investigated in subpopulations of ELF-153. A high level of GATA-1 and GATA-2 mRNA was only present in the CD61+ cells. Therefore, these two transactivating factors may play an important role in the MK differentiation of ELF-153. We conclude that ELF-153 might be an important tool to investigate the mechanisms by which transcription factors control differentiation of MK progenitors.

Megakaryocyte growth in vitro predicts outcome in idiopathic thrombocytopenic purpura.

The impact of megakaryocyte growth in vitro on clinical data, especially outcome, was studied in 25 consecutive children with idiopathic thrombocytopenic purpura (ITP). Twenty children with untreated de novo ITP and five children with pretreated ITP were evaluated. The number of megakaryocyte colonies (cloning efficiency), the mean cell number per colony (mitotic amplification) and the percentages of polyploid megakaryocytes after 7 and 12 days in culture (relative size of the endomitotic compartment) were determined in two separate clonal assays. The culture data were related to clinical findings and outcome of the thrombocytopenia. The mean cell number per megakaryocyte colony was significantly correlated with the observed increase in the platelet count 5 days after starting therapy (n = 23; r = 0.642), and a significant negative correlation was found between the relative size of the endomitotic compartment and the duration of thrombocytopenia after bone marrow culture analysis (n = 25; r = -0.503). If all 25 children with ITP (untreated de novo and pretreated ITP) were considered, a normal frequency of polyploid megakaryocytes was associated with a duration of ITP for < 6 months in 14 of 16 cases, whereas an impaired polyploidization predicted a persistence of ITP for > 6 months in 9 of 9 cases (p < 0.0005); if only children with untreated de novo ITP (n = 20) were considered, 13 of 15 children with a normal polyploidization had an acute course of their ITP and 5 of 5 children with an impaired polyploidization developed chronic ITP (p < 0.003). The results in this small group of patients suggest that the assessment of the relative size of the endomitotic compartment after 7 and 12 days in plasma clot culture actually appears to be the best method for predicting a chronic course in children with ITP.

Mode of Inheritance of the Higher Degree of Megakaryocyte Polyploidization in C3H Mice. I. Evidence for a Role of Genomic Imprinting in Megakaryocyte Polyploidy Determination

AbstractC3H mice have higher average ploidy megakaryocytes than all other mouse strains tested, but the mode of inheritance of this anomaly is unknown. Therefore, to clarify the genetics of high ploidy megakaryocytes in C3H mice, we measured megakaryocyte DNA content from both male and female offspring from F1, as well as backcross mat-ings. In all, offspring from seven different matings of mice were studied: (1) C57BL X C57BL (the first strain listed is the male parent in each case), (2) B6C3F1 (offspring from C57BL X C3H mating) X C57BL, (3) C57BL X B6C3F1, (4) C57BL X C3H, (5) C3H X B6C3F1, (6) B6C3F1 X C3H, and (7) C3H X C3H. The polyploid megakaryocyte DNA content distributions of the offspring from these matings show that C3H mice have higher percentages of high ploidy megakaryocytes than did all other mice. Also, male mice had significantly higher percentages of high ploidy (32N and 64N) megakaryocytes than did female mice for all matings, except backcross mating no. 6. The megakaryocyte DNA content for individual offspring of a given back-cross appeared to form a single, continuous distribution, rather than segregate into two distinct groups, suggesting that the higher megakaryocyte DNA content of C3H mice is caused by involvement of multiple allelles. This conclusion is further supported by our finding that the frequency of high ploidy megakaryocytes among offspring of the various matings was related to the proportion of C3H genotype contributed by the parents, ie, average megakaryocyte DNA content increased linearly(r2 = .88 for male mice and .84 for female mice, P < .0001) with increasing C3H gene dosage; the correlations for both male and female mice were essentially parallel (slope = 0.08 and 0.09, respectively). In addition, we found an effect of genomic imprinting on megakaryocyte DNA content in backcross offspring. The genetic imprinting was characterized by the female parent having a greater influence on the offspring’s megakaryocyte DNA> content than the male parent, ie, although the overall genetic makeup was the same, female offspring from backcross no. 6 (in which the female was C3H) had higher average megakaryocyte ploidy values than those from backcross no. 5 (in which the female was B6C3F1). The same phenomenon was observed when comparing the results from backcrosses no. 2 and 3, ie, male offspring from mating no. 3 (B6C3F1 female parent) had a higher average polyploid megakaryocyte DNA content than did male offspring from backcross no. 2 (C57BL female parent). Thus, we find that a number of factors influence megakaryocyte DNA content, including genomic imprinting and male sex hormones; however, we conclude that the higher megakaryocyte DNA content of C3H mice results from the interaction of multiple alleles that are additive. We hypothesize that a larger dosage of these genes in the C3H mouse is responsible for the higher DNA content of their megakaryocytes.

Mode of inheritance of the higher degree of megakaryocyte polyploidization in C3H mice. I. Evidence for a role of genomic imprinting in megakaryocyte polyploidy determination

C3H mice have higher average ploidy megakaryocytes than all other mouse strains tested, but the mode of inheritance of this anomaly is unknown. Therefore, to clarify the genetics of high ploidy megakaryocytes in C3H mice, we measured megakaryocyte DNA content from both male and female offspring from F1, as well as backcross matings. In all, offspring from seven different matings of mice were studied: (1) C57BL X C57BL (the first strain listed is the male parent in each case), (2) B6C3F1 (offspring from C57BL X C3H mating) X C57BL, (3) C57BL X B6C3F1, (4) C57BL X C3H, (5) C3H X B6C3F1, (6) B6C3F1 X C3H, and (7) C3H X C3H. The polyploid megakaryocyte DNA content distributions of the offspring from these matings show that C3H mice have higher percentages of high ploidy megakaryocytes than did all other mice. Also, male mice had significantly higher percentages of high ploidy (32N and 64N) megakaryocytes than did female mice for all matings, except backcross mating no. 6. The megakaryocyte DNA content for individual offspring of a given backcross appeared to form a single, continuous distribution, rather than segregate into two distinct groups, suggesting that the higher megakaryocyte DNA content of C3H mice is caused by involvement of multiple allelles. This conclusion is further supported by our finding that the frequency of high ploidy megakaryocytes among offspring of the various matings was related to the proportion of C3H genotype contributed by the parents, ie, average megakaryocyte DNA content increased linearly (r2 = .88 for male mice and .84 for female mice. P &lt; .0001) with increasing C3H gene dosage; the correlations for both male and female mice were essentially parallel (slope = 0.08 and 0.09, respectively). In addition, we found an effect of genomic imprinting on megakaryocyte DNA content in backcross offspring. The genetic imprinting was characterized by the female parent having a greater influence on the offspring's megakaryocyte DNA content than the male parent, ie, although the overall genetic makeup was the same, female offspring from backcross no. 6 (in which the female was C3H) had higher average megakaryocyte ploidy values than those from backcross no. 5 (in which the female was B6C3F1

Effects of the Recombinant Hematopoietic Growth Factors Interleukin-3, Interleukin-6, Stem Cell Factor, and Leukemia Inhibitory Factor on the Megakaryocytic Differentiation of CD34+ Cells

AbstractUsing a liquid culture system and human CD34+ marrow cells, we examined the effects of recombinant interleukin (IL)-3, IL-6, stem cell factor (SCF), and leukemia inhibitory factor (LIF) on megakaryocyte (MK) growth, endoreplication, and maturation. MK proliferation, ploidy distribution, and volume were studied by flow cytometry. IL-3 was the only cytokine that, alone, induced a marked increase in MK proliferation. At a high CD34+ cell concentration, addition of IL-6, SCF, and LIF to IL-3–containing medium increased the number of MK (–20%). At a low CD34+ cell concentration, IL-3 alone was a less potent inducer of MK growth, but IL-6, SCF, and their combination had a marked effect, increasing the number of MK by a factor 1.7, 2.9, and 4.4, respectively. These differences may be related to the endogenous release of cytokines in the culture. The effects of these cytokines were subsequently tested on a more mature type of MK progenitor (CD34+ cells isolated after 6 days of incubation in liquid culture). IL-3 remained the most potent cytokine, but IL-6 or SCF alone also increased MK number in comparison to unstimulated cultures. The ploidy distribution of MKs grown with IL-3 was not markedly changed by the addition of the other cytokines, with the exception of SCF, which induced a significant increase in the mean ploidy. However, in all cultures, glycoprotein (GP)IIIa + 2N and 4N cells were present in large but variable numbers (35% to 75%). The number of these low-ploidy MKs directly correlated with MK proliferation. Therefore, we subsequently explored the absolute number of polyploid MK produced in culture. SCF, IL-6, or their combination, in association with IL-3, increased the number of polyploid MK up to fourfold. In addition, they improved the maturation of MK grown in the presence of IL-3, leading to the synthesis of demarcation membranes and platelet shedding. A similar effect of growth factors on the maturation of day 6 CD34+ cells was observed. We conclude that IL-6 and SCF have a broad range of activities on megakaryocytopoiesis, acting both on the early and late stages. However, the proliferative properties of these cytokines largely predominate in our cultures. Therefore, in the absence of a specific MK regulator, this study further extends the need for a combination of growth factors to maximize megakaryocytopoiesis.

The regulation of megakaryocyte polyploidization and its implications for coronary artery occlusion

Polyploidization is a distinctive feature of megakaryocyte differentiation. The physiological meaning and the regulation of this process are obscure. Megakaryocyte ploidy varies in normal biology and in disease. Here we review the evidence suggesting that ploidy changes may have a role in the determination of platelet reactivity and in the aetiology of coronary artery occlusion. We also present a hypothesis that may serve as a framework to explore the regulation of megakaryocyte polyploidization at the molecular level and also may provide a rational basis to explain the occurrence of ploidy changes in ischaemic heart disease.

Expression of CD34 and platelet glycoproteins during human megakaryocytic differentiation

Megakaryocyte (MK) progenitors express the CD34 antigen, but the precise stage along the MK differentiation at which the CD34 is turned off is not known. Purified marrow CD34+ cells give rise within 4 days in culture to rare mature MK, suggesting that some MK precursors bear the CD34 antigen. By multiparameter flow cytometry, CD34+ cells bearing platelet glycoproteins (GP) could be detected, but at a low frequency (less than 2% of the marrow CD34+ cells). We used an in vitro liquid suspension culture to selectively amplify MK differentiation. CD34+ cells were isolated after 6 days before a wave of mature MK. These cells gave rise within another 4 days in culture to numerous MK (up to 50%), showing that these CD34+ cells were greatly enriched in MK precursors. This was confirmed by ultrastructural studies that showed the presence of typical promegakaryoblasts. By flow cytometry, three populations of small cell size could be defined: CD34+ GPIIIa-, CD34+ GPIIIa+, and CD34- GPIIIa+ cells. The two GPIIIa+ populations were almost pure immature blastic MK. alpha-Granules were rare in the CD34+ GPIIIa+ cells, whereas they were more developed in the CD34- GPIIIa+ cells, which also exhibited demarcation membranes. Approximately 45% of the two GPIIIa+ cell populations were capable of undergoing at least one cell division and of giving rise to a polyploid progeny. However, proliferation and polyploidization capacities were higher in the CD34+ GPIIIa+ than in the CD34- GPIIIa+ cells. A small fraction of GPIIIa+ cells (about 10%) were able to give rise to MK colonies containing a maximum of 16 cells for the double-positive cells. GPIb was expressed on about sixfold less cells than GPIIIa, but was detected on a few CD34+ cells. Most double-stained (CD34+ GPIb+) cells were polyploid. CD34- GP+ cells (more mature) contained less polyploid MK than the CD34+ GP+ fraction. Altogether, these findings show that CD34 is still expressed on a polyploid transitional immature MK and that GPIIIa is present on some MK progenitors with low proliferative capacities. They also suggest that the expression of CD34 is related to the ability of the MK precursors to accomplish DNA synthesis (either cell division or endomitosis). Such a characterization will facilitate the investigation of the role of the different cytokines on MK differentiation.

Expression of CD34 and Platelet Glycoproteins During Human Megakaryocytic Differentiation

Megakaryocyte (MK) progenitors express the CD34 antigen, but the precise stage along the MK differentiation at which the CD34 is turned off is not known. Purified marrow CD34+ cells give rise within 4 days in culture to rare mature MK, suggesting that some MK precursors bear the CD34 antigen. By multiparameter flow cytometry, CD34+ cells bearing platelet glycoproteins (GP) could be detected, but at a low frequency (less than 2% of the marrow CD34+ cells). We used an in vitro liquid suspension culture to selectively amplify MK differentiation. CD34+ cells were isolated after 6 days before a wave of mature MK. These cells gave rise within another 4 days in culture to numerous MK (up to 50%), showing that these CD34+ cells were greatly enriched in MK precursors. This was confirmed by ultrastructural studies that showed the presence of typical promegakaryoblasts. By flow cytometry, three populations of small cell size could be defined: CD34+ GPIIIa-, CD34+ GPIIIa+, and CD34- GPIIIa+ cells. The two GPIIIa+ populations were almost pure immature blastic MK. alpha-Granules were rare in the CD34+ GPIIIa+ cells, whereas they were more developed in the CD34- GPIIIa+ cells, which also exhibited demarcation membranes. Approximately 45% of the two GPIIIa+ cell populations were capable of undergoing at least one cell division and of giving rise to a polyploid progeny. However, proliferation and polyploidization capacities were higher in the CD34+ GPIIIa+ than in the CD34- GPIIIa+ cells. A small fraction of GPIIIa+ cells (about 10%) were able to give rise to MK colonies containing a maximum of 16 cells for the double-positive cells. GPIb was expressed on about sixfold less cells than GPIIIa, but was detected on a few CD34+ cells. Most double-stained (CD34+ GPIb+) cells were polyploid. CD34- GP+ cells (more mature) contained less polyploid MK than the CD34+ GP+ fraction. Altogether, these findings show that CD34 is still expressed on a polyploid transitional immature MK and that GPIIIa is present on some MK progenitors with low proliferative capacities. They also suggest that the expression of CD34 is related to the ability of the MK precursors to accomplish DNA synthesis (either cell division or endomitosis). Such a characterization will facilitate the investigation of the role of the different cytokines on MK differentiation.

Megakaryocyte polyploidy and maturation in chronic granulocytic leukemia.

To understand abnormal platelet production in chronic granulocytic leukemia, polyploidization and maturation of megakaryocytes in 10 patients were studied using a technique which allows sequential immunofluorescence identification by a monoclonal platelet antibody (C17), cytophotometric determination of the relative DNA content and cytological characterization of megakaryocytes in panoptically stained smears. Compared to normal conditions the proportion of diploid promegakaryocytes was not increased, suggesting an undisturbed influx of progenitor cells into the megakaryocytic cell compartment. Small tetraploid (4c) megakaryocytes undergo maturation without further polyploidization, the so-called microkaryocytes being mature rather than immature cells. Most of the megakaryocytes show rhythmical polyploidization only up to octoploid (8c) level, indicating the inability to produce high-polyploidy cells.

Exogenous and endogenous regulations of human megakaryocytopoiesis.

Megakaryocytopoiesis may be viewed as a developmental continuum which begins when an undifferentiated hematopoietic stem cell commits to maturation within this lineage (for recent review see ref.#l) The process of commitment results in the generation of megakaryocyte progenitor cells (CFU-Meg). When appropriately stimulated, CFU-Meg are capable of intense proliferative activity which determines the numbers of megakaryocytes which will ultimately populate the bone marrow. As proliferative activity declines, CFU-Meg progeny mature into precursor cells which terminally differentiate into polyploid megakaryocytes capable of releasing several thousand platelets each into the circulation.

Significance of polyploidy in megakaryocytes and other cells in health and tumor disease.

Polyploidy--the doubling of chromosome sets of cells caused by a stop of mitosis at different levels of the mitotic cycle--is a phenomenon widely observed in plants, protozoa, metazoa, and animals. In man obligate polyploid tissues are found in liver parenchyma, heart muscle cells, and bone marrow megakaryocytes. Polyploidy occurs mostly in stable and highly differentiated cells and tissues. Besides age, stimulation of proliferation and increased metabolic function lead to polyploidization in these organs. Aneuploidy, however, is exclusively found in tumor cells. Megakaryocyte differentiation and polyploidy are controlled by thrombopoietin-like activities, of which the loci of production are still unknown. Megakaryocytes are unique among polyploid mammal cells. On the precursor level they maintain their proliferative activity independently of the mammal's age. Once having entered the incomplete mitotic cycle they stop cytokinesis and develop into highly polyploid cells. Polyploidization of megakaryocytes is the basic requirement for establishing highly effective hemostasis in mammals, which exhibit blood circulation based on high blood pressures. Every polyploidization results in increased production of membrane materials with which the platelet becomes endowed. By shedding cytoplasmic fragments approximately 3000 platelets are set free from a 32c megakaryocyte, compared with only 16 nucleated thrombocytes by mitotic division. There is further evidence that the heterogeneity of platelets mostly depends on the different polyploidy classes of the megakaryocytes from which they are derived. Changes in the polyploidy pattern of megakaryocytes could therefore have consequences for hemostatic disorders in several human diseases, particularly in malignancy.

An analysis of the platelet and polyploid megakaryocyte response to acute thrombocytopenia and its biological implications.

Thrombocytopenia was induced in rabbit by a single injection of anti-platelet serum. The circulating platelet number and concomitant mean platelet volume were measured 6 h after the injection and daily thereafter. The megakaryocyte nuclear DNA content was measured in control rabbits and after 2 h and 24 h of thrombocytopenia respectively. The observed recovery of circulating platelet number is used to construct the variation of platelet destruction rate and platelet production rate with time during thrombocytopenia. The observed rebound thrombocytosis is explained by using a balance equation of these two rates. Platelet production is linked to the megakaryocyte by physical fragmentation theory and an explanation of the observed variation of mean platelet volume is used to obtain an estimated bleeding time and circulating platelet biomass during recovery from thrombocytopenia. An interpretation is given of the homeostatic control of megakaryocytopoiesis and thrombopoiesis.

Polyploid megakaryocytes develop randomly from a multicompartmental system of committed progenitors.

Cumulative distributions of the number of doublings undergone by mixed megakaryocytic/erythroblastic colonies and by pure megakaryocytic colonies were determined from plasma clot cultures of bone marrow (from C57BL/6 mice) supplemented with erythropoietin. Analysis of these distributions suggests that these colonies are produced by three distinct progenitors. At days 7-14, progenitors of mixed megakaryocytic/erythroblastic colonies (BFU-ME) generate tri-exponential distributions and the mean (+/- SD) fraction of this progenitor pool ceasing to proliferate per doubling (FCP) increases stepwise from 0.07 +/- 0.06 to 0.27 +/- 0.07 and 0.73 +/- 0.07. In this interval, progenitors of pure megakaryocytic colonies (CFU-M) generate bi-exponential slopes whose FCP values are compatible with the two last slopes above. Finally, CFU-M at day 3 express only the last slope. From days 5 to 9, megakaryocytes generated by BFU-ME reach lower ploidy levels than do those generated by CFU-M. It is concluded that, in the culture system used, (i) megakaryocyte progenitors that do not switch to polyploidization mature through the three consecutive compartments indicated, (ii) each progenitor population has a probability of becoming polyploid that reflects the fraction that ceases to proliferate, (iii) the exponentially distributed mitotic reserve of progenitors is determined by the combination of maturing into the next compartment and the probabilistic switch to the pathway of polyploidization, and (iv) the ploidy distribution of megakaryocytes probably depends on the progenitor from which they originate.

Megakaryocyte polyploidization in May-Hegglin anomaly.

Polyploidization of megakaryocytes was studied in bone marrow aspirates from 3 patients with May-Hegglin anomaly by combined application of cytophotometric determination of the DNA content and autoradiography with 3H-TdR labeling in vitro. A marked elevation of the influx of progenitor cells into the megakaryocytic cell system as well as a decreased maturation capacity from type II to type III megakaryocytes was observed possibly contributing to the pathological platelet sequestration. The polyploidization activity as assessed by 3H-TdR labeling and nuclear DNA content was normal.

Megakaryocyte Polyploidization in Myeloproliferative Disorders

Megakaryocyte Polyploidization in Myeloproliferative Disorders

Megakaryocyte polyploidization in acute leukaemia and preleukaemia.

Summary. Megakaryocyte polyploidy was studied in six cases of preleukaemic acute leukaemia (PL), and in acute leukaemia (AL), five cases in the overt phase and three during complete remission. The megakaryocyte polyploidization was determined cytophotometrically and in some cases also by in vitro labelling with tritiated thymidine (3H‐TdR). The results obtained in PL and untreated AL. demonstrate an impairment of the 3H‐TdR labelling index and altered megakaryocyte polyploidy such as decrease of the maximum polyploidization capacity (five cases), evidence of diploid megakaryocytes (two cases) and of hyperploid megakaryocytes (six cases). These disturbances reflected the cytological abnormalities (microkaryo‐cytes, polykaryocytes) observed in most of the cases studied.In contrast, in cases studied during complete remission of AL. a normal labelling index and polyploidy composition was observed, suggesting that the polyploidization disturbances are reversible. A strict correlation of the polyploidization defect and the platelet production could not be established from the patients investigated.

Polyploidization of megakaryocytes in normal humans, in patients with idiopathic thrombocytopenia and with pernicious anaemia.

Summary. Polyploidization of human megakaryocytes was studied in bone marrow aspirates from three normal individuals, three patients with idiopathic thrombocytopenic purpura (ITP) and three patients with pernicious anaemia (PA). The cells were labelled in vitro with tritiated thymidine and analysed by a combined cytophotometric and autoradiographic method. Cells were divided into types I, II and III.Normal megakaryocytes showed ploidy stages from 4c to 32c, the 4c stage occurring only in type I. The labelling indices showed that in normal megakaryocytes polyploidization takes place in type I and II and not in type III cells. This study provided evidence for alternation of DNA synthesis and rest periods. It is postulated that rhythmical polyploidization in megakaryocytopoiesis takes place. The progressive increase in the proportion of cells in successive resting periods (G‐phases) suggests that high ploidy cells have longer resting periods than low ploidy cells.In ITP, megakaryocyte morphology, labelling indices and distribution of DNA content were normal. Assuming a normal ploidy composition in ITP, the megakaryocytic hyperplasia observed in typical cases may be explained by an increased influx from the unrecognizable progenitor cell.In PA, an arrest of megakaryocytic DNA synthesis was observed comparable to that found in megaloblastic erythropoiesis.

Formation of splenic megakaryocytes in young rat

The formation of the giant polyploid megakaryocyte was followed in the spleen of the 3 week old rat using 3H-thymidine. There is random aggregation and synchronous fusion of precursor cells (probably lymphocytes) which have completed DNA synthesis. These form a multinucleated cell which is in the G2 phase of the cell cycle. The nuclei of the multinucleated cell then enter mitosis synchronously and progress through metaphase. At this time the multipolar chromosomal groups fuse and form the polyploid nucleus of the mature megakaryocytes. The multinucleated stage is shortlived but may be delayed as long as 14 h in its transit to the giant polyploid stage by the internal radiation from a 10 μCi/g labeling dose of 3H-thymidine.

Maturation of rat megakaryocytes studied by microspectrophotometric measurement of DNA.

SummaryThe amount of DNA in the nuclei of individual megakaryocytes of male Sprague-Dawley rats was measured micro-spectrophotometrically in marrow smears stained by the Feulgen reaction. When the DNA values were arranged in a frequency distribution, the megakaryocytes fell into groups with the modal DNA content of successive groups increasing approximately by a factor of 2. Ploidy of the megakaryocyte groups was determined by comparison with DNA values of diploid cells. Frequencies of 4N, 8N, 16N, and 32N megakaryocytes were, respectively, 1.6%, 10.5%, 71.2%, and 17.1%. The results indicate synchronous division of the nucleus of polyploid megakaryocytes. The relative numbers of cells in ploidy stages may indicate the relative length of time spent in the stages. An hypothesis of megakaryocyte maturation is proposed. The method may prove useful in studies of megakaryocyte maturation and regulation.ADDENDUM: In a recent microspectrophotometric study, de Leval observed a frequency distribution pattern of DNA...