Polyploid megakaryocytes develop randomly from a multicompartmental system of committed progenitors

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Proc. NatL Acad. Sci. USA Vol. 79, pp. 4410-4414, July 1982 Medical Sciences

Polyploid megakaryocytes develop randomly from a

multicompartmental system of committed progenitors

(bone marrow cultures/two-color microspectrophotometry/colony size distribution/age structure-probabilistic model)

JEAN-MICHEL PAULUS*t, MICHEL PRENANT*, JEANNE-FRAN5OISE DESCHAMPS*t, AND MICHEL HENRY-AMAR§ *Institut de Pathologie Cellulaire (INSERM U48 and Association Claude Bernard), HMpital de Bicktre, 94270 Kremlin-Bicktre, France; tD6partement de Clinique et sur Thrombose Experimentale et l'Hemostase (INSERM U150), MHpital Pathologie MWdicales, Universit6 de Liege, 4020 Liage, Belgium; WUnitM de Recherches la Lariboisiere, 75475, Paris C~dex 10, France; and §Unit6 de Recherche Statistique (INSERM U21), 16 bis Avenue Paul Vaillant-Couturier, 94800 Villejuif, France

Communicated by Eugene P. Cronkite, March 15, 1982

cells which polyploidize without. first dividing (18). AcChoEase is secreted by megakaryocytes and may play a role in the regulation of thrombocytopoiesis (19). The mitotic amplification undergone by megakaryocyte progenitors in culture can be quantified from the number of doublings required to achieve the measured colony size. Similarly, polyploidization can be expressed as the number of endoduplications producing the measured megakaryocyte ploidy. Therefore, megakaryocytic colonies offer the opportunity to relate early and. late amplification quantitatively at two distinct stages of clonal development. We show here that maturation of megakaryocyte progenitors is associated with abrupt increases in the probability -of arrest of mitotic. proliferation and. switch to polyploidization and that at least three megakaryocyte progenitor compartments can be identified, each being characterized by an exponential distribution of the number of doublings undergone before initiating polyploidization.

ABSTRACT 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 ofthese 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 o.27 ± 0.07 and to 0.73 ± 0.07. In this interval, progenitors of pure megakaryocytic colonies (CFU-M) generate biexponential 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, (Q) each progenitor population has. a probability of becoming polyploid that reflects the fraction that ceases to proliferates (i#i) 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.

Nuclear amplification in the. mammalian thrombocytic series can be shown to develop in two steps in bone marrow cultures. The first step consists of a wave of mitotic divisions undergone by an unrecognized progenitor which, in cultures of murine bone marrow, produces clones composed of pure megakaryocytes (1-6), megakaryocytes and erythroblasts (7.-s), or several lineages besides megakaryocytes (10-13). The clonal origin of the mixed colonies has been demonstrated by single-cell transfer experiments.(12) and by chromosomal analysis (8). The second nuclear amplification step does not yield any increase in cell number but rather consists of a wave of endoduplications resulting in polyploid megakaryoblasts and megakaryocytes. The distribution of DNA content in non-DNA-synthesizing megakaryocytes ranges from 2 to 64 times the haploid value and has modes at successive powers of 2N (14-17). This sequence of endoduplications is termed "polyploidization." In mice, commitment to polyploidization is marked by the synthesis of ac-

MATERIALS AND METHODS Cell Cultures. Femoral bone marrow from C57BL/6 mice (6-8 wk old) was cultured at the concentration of 30,000 nucleated cells per ml of plasma clot medium according to McLeod et aL (20), except that alpha medium (Flow Laboratories, McLean, VA) was substituted for NCTC and the bottom of 35i mm Petri dishes was covered with a .33-mm coverslip on which the culture suspension was layered. Interposition of the glass coverslip prevented melting of the plastic Petri dishes when the preparations were placed under immersion oil during the lengthy microspectrophotometric measurements of DNA (see below). Formation of erythroid and megakaryocytic colonies was stimulated by including erythropoietin (Epo) in the medium. Qualitatively similar results were obtained with serum from anemic mice (21) used at 50 gl/ml, and two Epo preparations [human. urinary Epo, obtained from Ann Ball (National Institutes of Health, Bethesda, MD) in experiments 2-5, and step III preparation from sheep plasma.(Connaught, Toronto, ON) in experiment 6], both used at the plateau concentration of 3 units/ml. Megakaryocyte Identification and Measurement of Ploidy. After 3-14 days the coverslips and the adherent plasma clots were double stained for AcChoEase and DNA. The preparations.were fixed for 10 min in 5% glutaraldehyde in phosphate

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Abbreviations: CFU-M, progenitor of pure megakaryocytic colonies; BFU-ME, progenitor of mixed megakaryocytic/erythroblastic colonies; AcChoEase, acetylcholinesterase; Epo, erytropoietin; FCP, fraction of progenitors ceasing to proliferate in one doubling interval.

etylcholinesterase (AcChoEase) in small, presumably diploid


Medical Sciences: Paulus et aL

Proc. Natd Acad. Sci. USA 79 (1982)

buffer at pH 7.0, rinsed three times for 1 hr and then three times for 1 day in buffer, and stained for AcChoEase according to Jackson (22) but without the postfixation in methanol and counterstaining with hematoxylin. After drying at 37TC the coverslips were stained with the Feulgen reaction (23); acid hydrolysis was with 3.5 M HCI at 250C for 1.5 hr. After dehydration the preparations were mounted on a glass slide. Megakaryocytes were recognized by the presence of at least one localized deposit of AcChoEase reaction product; erythroblasts, macrophages, and granulocytes had no such precipitate and were distinguished on the basis of the shape and pattern of their Feulgen-stained nucleus. Megakaryocyte ploidy determination was adapted from Paulus (17) and was based on scanning microphotometry with the Zeiss Axiomat (Oberkochen, Federal Republic of Germany) operated successively at 560 and 450 nm. This wavelength combination allowed accurate separation of the absorbances due to DNA and AcChoEase reaction products. The specific Feulgen extinction at 560 nm was calculated from F5w = (Ewo kE4) [m/(m - k)] in which Ew and E4w are the total measured extinctions at 560 and 450 nm, respectively. The value k, which averaged 0.56 0.07 (± SD), was the ratio of cytoplasmic extinction at 560 and 450 nm for the AcChoEase-associated precipitate; m, which averaged 1.98 0.16, was the corresponding value for the Feulgen stain, measured in granulocyte nuclei. Ploidy was the ratio of F5w and the half-diploid value obtained on mature granulocytes. A total of 829 megakaryocytes in 168 colonies were evaluated for ploidy in this study, in addition to a total of 512 megakaryocytes reported in a preliminary work (24). Characterization of Megakaryocyte Colonies. The x-y location of each megakaryocyte was recorded at X250 magnification and plotted at 10:1 scale on a circular millimeter paper. At the low cell concentration used, colonies were generally well separated (Fig. 1 Left) and visual inspection of the plots made at low concentration (10,000 cells per plate) indicated that, between days 5 and 9, no colony cell had wandered further than 1,000 ,um (700 ,um for 3-day colonies) from the nearest colony member. This distance was then used to delineate colonies in more concentrated cultures. In addition to cellular composition,


each colony was characterized by the number of doublings undergone by its progenitor in the megakaryocyte pathway-i.e., log2 of the number of AcChoEase-positive cells per colony. This identification criterion was chosen because such cells are essentially nondividing precursors committed to polyploidization (18) and because, after 5-14 days of culture, pure colonies contained
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