British Journal of Haernatologg, 1982, 52, 173-180

Annotation

THE ORIGIN, DEVELOPMENT AND REGULATION OF MEGAKARYOCYTES

Much insight into platelet production and function has been gained by examination of megakaryocytes and their precursors. Since the last major reviews on this subject (Ebbe, 19 76; Breton-Gorius & Reyes, 19 76). significant advances have occurred in our knowledge of megakaryocytopoiesis. This field was recently stimulated by the first conference devoted to the in vitro study of megakaryocytes (Evatt et al, 1981). This Annotation considers the progressive differentiation of marrow stem cells into platelets and the regulatory activities involved therein.

Introduction

Platelets are enucleate lblood cells which are formed from megakaryocytes. During development and maturation, megakaryocytes form probably all platelet components save serotonin (Nachman et al, 1977; Rabellino et al, 1979: Chernoff et aI, 1980; Schick & Weinstein, 1981). They achieve a variable degree of polyploidy (Mayer et al, 1978: Levine et al, 1980) in the course of (development. Megakaryocytes have now been shown to originate from undifferentiated progenitors (Metcalf et al, 1975). A spectrum of those precursors can be stimulated in vitro to form colonies (Williams & Jackson, 1978; Levin et al, 1981). Megakaryocyte colony development is usually restricted to a single lineage, although colonies of two or more cell types, megakaryocytes with granulocytes, erythroid cells and/or macrophages, have been reported (Fauser & Messner, 1979; Metcalf et al, 1979).

Megakaryocyte characterization and development

Table I illustrates the different maturation stages of megakaryocytes, including size ranges, and changes in nuclear configuration. These characteristics allow appreciation of the full spectrum of megakaryocyte development, from the beginning of differentiation to the shedding of platelets at maturity (Levine, 1980; Levine et al, 1982). Many more megakaryocytes can now be recognized by the use of this more precise cytological characterization. These new criteria have allowed an additional 20-30% of megakaryocytes to be identified, mostly smaller than 20 pm in diameter. The smallest, youngest megakaryocytes detected have a diameter of 10 pm or less (MacPherson, 1971; Jackson, 1973; Levine, 1980: Rabellino et al, 1981; Levine et al, 1982). On marrow smears, many quite immature megaka:ryocytes are already larger than all but a very few of the nonmegakaryocytes (Levine et al, 1982). In addition to this size threshold, the youngest

Correspondence: Dr Richard F. Levine, VA Medical Center, 50 Irving St., N.W., Washington, D.C. 20422, U.S.A.

0007-1048/82/1000-0173!~02.00 @ 1982 Blackwell Scientific Publications 171

Tabl

e I. C

ytol

ogic

cha

ract

eris

tics o

f meg

akar

yocy

te m

atur

atio

n st

ages

. In

the

first

col

umn

are

diag

ram

s of

the

nuc

lear

con

figur

atio

ns

and

nucl

ear:

cyto

plas

mic

ratio

s at e

ach

stag

e. M

uch

of th

e su

e va

riatio

n fo

r eac

h st

age i

s rel

ated

to d

iffer

ence

s in

ploi

dy le

vel (

Levi

ne et

al

, 198

2). T

he li

kely

plo

idy

leve

ls of

eac

h st

age

are

sugg

este

d in

Fig

1.

Nuc

lear

C

ytop

lasm

ic st

aini

ng

App

roxi

mat

e D

emar

catio

n Su

gges

ted

Stag

e m

orph

olog

y (W

right

-Gie

msa

) siz

e ra

nge

mem

bran

es

Gra

nule

s na

me

I C

ompa

ct

Bas

ophi

lic

6-24

pm

Pr

esen

t by

Few

pre

sent

M

egak

aryo

blas

t (lo

bed)

el

ectro

n by

ele

ctro

n m

icro

scop

y m

icro

scop

y

I1

Hor

sesh

oe

Pink

cen

tre

14-3

0 p

n

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ifera

ting

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ting

to

Prom

egak

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cyte

to

cen

tre

of c

ell

incr

ease

Gra

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r I11

M

ultil

obed

In

crea

sing

ly m

ore

16-5

6 pm

Ex

tens

ive

but

Gre

at n

umbe

rs

pink

tha

n bl

ue

assy

met

ric

meg

akar

yocy

te

IV

Com

pact

but

W

holly

20

-50

pn

Ev

enly

O

rgan

ized

into

M

atur

e hi

ghly

lobu

late

d eo

sino

phili

c di

strib

uted

‘p

late

let f

ield

s’

meg

akar

yocy

te

Annotation 175

recognizable megakaryocytes have multiple overlapping nuclear lobes and may have a very small amount of deep blue cytoplasm (MacPherson, 1971; Levine, 1980; Levine et al, 1982) or, in the rodent, small amounts of acetylcholine esterase (stage I; Jackson, 1973).

With maturation the cell enlarges, acquires increasing amounts of cytoplasmic organelles while losing its basophilia, and the nuclear lobes disperse (stages I1 and 111; MacPherson, 1971; Levine et al, 1982). 'The fully mature megakaryocyte normally ranges up to 50 pm or so and has a tightly packed nucleus (stage IV; Levine, 19 7 7). The proliferation of invaginated surface membranes that iiltimately divide the megakaryocyte cytoplasm into individual platelets (demarcation membranes) begins in the youngest recognizable megakaryocytes (MacPherson, 1971; Levine, 1980), but actual liberation of platelets appears to occur simultaneously only at the end of the maturation process (Levine, 19 7 7). From ultrastruc- tural studies, it appears that perhaps 3000 platelets are liberated from each megakaryocyte (Chernoff et al, 1980). However, the actual number might be variable, depending on the final DNA level achieved in each megakaryocyte.

Because they form platelets, megakaryocytes may be defined by, among other attributes, the presence of platelet components or functions such as specific antigens (factor VIII antigen, various membrane antigens; Nachman et al, 19 7 7; Rabellino et al, 19 79) acetylcholine esterase (in certain animal species, Jackson, 19 73), or serotonin uptake (Schick & Weinstein, 198 1). Examination for platelet markers allows easy detection of megakaryocytes, especially the youngest and smallest, compared to traditional morphologic criteria (Rabellino et al, 1981). However, complete detection is not always obtained (Rabellino et al, 1979) and specificities remain to be confirmed.

Polyploidy and thrombopoie tin

During differentiation and maturation megakaryocytes progressively increase their DNA content. The approximate proportions at each level may be seen in Fig 1, although the frequencies for 2N and 4N megakaryocytes are only estimates (Mayer et al, 1978; Levine, 1980). The 8N megakaryocytes actually comprise a majority of the megakaryocyte population and were often overlooked in earlier studies (Levine et al, 1980).

Thrombopoietin is a substance(s) in the blood of thrombocytopenic subjects which is probably responsible for the achievement of higher ploidy levels in megakaryocytes (Ode11 et al, 1976). This hormone has been quantitated by its ability to stimulate incorporation of radioisotopes into newly produced platelets in recipient animals (Evatt & Levin, 1969). The level of thrombopoietin apparently varies inversely with the circulating platelet mass (Odell & Murphy, 1974). When platelets are normal or increased in number, the amount of thrombopoietin probably decreases, leading to a parallel fall in the average ploidy levels and size. As suggested in Fig 1, in normals most of the 8N and lower ploidy megakaryocytes are still immature (stages I and II; Odell et al, 1970; Levine ef al, 1982), whereas most of the 16N and higher cells are maturing (stage 111) or are fully mature (stage IV). Thrombopoietin might therefore be necessary for and sustain cytologic maturation of megakaryocytes (Long et al, 1982; Williams et aI, 19182). although this idea is not yet proven. Megakaryocyte size correlates with ploidy level or with maturational stage, so that higher ploidy cells of the same

176

Megakaryocyte I I Maturation II

IV Stages 111

Annotation

? ? I

Thrombopoietin - - - - - - - - L - . r Abnormalities1 Platelet Mass

1 Stimuli

? I Feedback Cell Cycle Inhibition -

7 Multipotential Stem Cells ,

Stimulus

M e g a k a r y o c y t e s I t l 2N 4N : 2N 4N 8N 16N 32N 64N 128N

Fig 1. Megakaryocytopoiesis. Shown horizontally in the centre is the progression from uncommitted multipotential stem cells, through the proliferating precursors detected in the in vitro cloning assays, to the spectrum of maturing megakaryocytes. The relative DNA levels of megakaryocytes and their precursors is given as ploidy values (N) where 2N is a diploid cell. The megakaryocytes are also represented vertically in terms of their maturation stages, ending in platelet shedding: a detailed classification of these stages is given in Table I. The columns show the maturation stages at particular ploidy ranges. Thus, 4N megakaryocytes are found at maturation stages I and 11. The top row shows a postulated two-level regulatory process with megakaryocyte colony stimulating factor (Mega-CSF) primarily influencing proliferation of the clonable precursors and thrombopoietin required for megakaryocyte ploidy amplification and possibly for maturation. It is not certain that these regulators are completely exclusive in their target cell specificities as shown. Furthermore, no clear distinction is currently possible between the specific or nonspecific control of the two factors. Thrombopoietin production is sensitive to variations in platelet mass, as indicated in the figure; it is assumed that this feedback mechanism operates on the source of thrombopoietin. The progenitors might be controlled by cell cycle inhibition, perhaps as a consequence of normal numbers of megakaryocytes.

stage have greater diameters than lower ploidy cells, and more mature cells are likewise larger than the less mature cells of the same ploidy level (Odell et ul, 1970,1976; Levine et al, 1982). It is believed that megakaryocytes with larger cytoplasmic volumes produce a greater number of platelets (Odell et al. 1976). although direct proof is still lacking. Thus, thrombopoietin amplifies platelet production by stimulating development of individual megakaryocytes.

Megakuryocyte precursors

Polyploidy in megakaryocytes appears to be achieved without a cell division following each

Annotation 177

mitosis. There is no evidence in vivo or in short-term primary culture that recognizable megakaryocytes can divide, i.e. undergo cytokinesis (Ebbe & Stohlman, 1965: Levine, 19 77). Increases in numbers of megakaryocytes therefore appear to be due to proliferation of some unrecognizable precursor. However, the possibility that newly differentiated, non-polyploid (i.e. 2N-4N) megakaryocytes (Mayer et al. 1978) can divide has not been disproven. Proliferation of megakaryocyte progenitors into colonies is stimulated by a substance(s), so far studied only in vitro, called megakaryocyte colony stimulating factor (Metcalf et al, 1975). This activity does not appear in the blood of acutely thrombocytopenic animals and therefore does not appear to be thrombopoietin (Levin et al, 1981). Thrombopoietin preparations appear to be unable to stimulate clonal proliferation (Williams et al, 1979: Hoffman et al, 198 1). Thus, a second megakaryocyte stimulatory factor exists, distinct from thrombo- poietin.

Two factor regulation

Two factors, therefore, appear to regulate megakaryocytopoiesis, affecting proliferation and development separately in vitro (Fig 1). In this scheme, megakaryocyte colony stimulating factor stimulates population expansion by cell division of the progenitor cells, while thrombopoietin influences megakaryocyte development. Additive studies show that both factors are necessary for full colony development (Williams et al, 19 79; Hoffman et al, 198 1). No colonies of recognizable megakaryocytes are observed when megakaryocyte colony stimulating factor is used as the sole stimulus in vitro (Williams et al, 1982).

The specificity of the various colony stimulating factors in vivo remains uncertain. Increases in both myeloid and megakaryocyte progenitors are observed in thrombocytopenic animals (Levin et al, 1980). The converse is also observed in vivo: haemolytic anaemia, iron deficiency, and serious infection may be accompanied by thrombocytosis. Megakaryocyte colony stimulating factor is difficult to detect in vivo; demonstrable levels have been reported in urine (Enomoto et al, 1980) or serum (Hoffman et al, 1981) for aplastic anaemia patients. Normal bone and lung contain very small amounts (Williams et al, 1981).

In vivo studies also support a bi-level regulation of megakaryocyte formation. In acutely thrombocytopenic animals, increased numbers of high ploidy and large megakaryocytes are observed. with no immediate change in megakaryocyte progenitor numbers (Levin et al, 1980). Kidney cell thrombopoietin, certain cell and tissue derived conditioned media (Williams et al, 1979, 19111) and thrombocytopenic animal plasma (Levin et al, 1980) have little or no capacity by themselves to stimulate colony formation in vitro. However, they are required for the increase in size and acetylcholine esterase content of isolated individual immature megakaryocytes and increases in the DNA content of megakaryocytes in developing colonies (Long et al, 1982; Williams et ul, 1982). As noted, serum extracts from most thrombocytopenic human patients (i.e. thrombopoietin) will not stimulate colony formation in vitro: serum from aplastic anaemia-patients does, however, and apparently may have both activities, i.e. the megakaryocyte colony stimulating factor and thrombopoietin.

Feedback regulation may also occur at these two levels. An increased platelet mass inhibits the formation and development of megakaryocytes. Megakaryocyte numbers are

178 Annotation suppressed only partially by thrombocytosis, by one-quarter to two-thirds at most. Thus, it appears that thrombopoietin cannot be completely inhibited, and/or that the number of megakaryocytes is not much affected by thrombopoietin. The delayed increase of the progenitor cells after thrombocytopenia also suggests a separate control mechanism. The actual mechanism is unknown: since the clonable precursors are non-cycling in vivo (Williams & Jackson, 1978). response to changes in megakaryocyte numbers is a possible mechanism (Hoffman et al, 1981).

Two other possible control points in megakaryocytopoiesis can be considered, but there is scarcely any information available on them: the control of the rate of commitment of multipotential stem cells into megakaryocytic precursors and the initiation of megakaryo- cytic differentiation from the precursors.

Conclusion

The better characterization of young megakaryocytes, the identification of the clonable progenitor pool, and the recognition of a bilevel regulation are recent, significant contributions to our understanding of platelet production. We now have a better defined operational framework with which to ask questions about important clinical platelet disorders.

ACKNOWLEDGMENTS

Supported by U.S.P.H.S. grants HL 22451, HL 23399 and CA 08748, by American Cancer Society grant IN-114, and by the Veterans Administration. N.W. is a Leukemia Society of America Scholar.

Sloan-Kettering lnstitute for Cancer Research, Rye, N.Y., and V A Medical Center, Washington, D.C., U.S.A.

NEIL WILLIAMS RICHARD F. LEVINE

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Annotation 179 FAUSER. A.A. & MESSNER, H A . (1979) Identifica-

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37,265-270.

775-782.

765-775.

180 Annotation WILLIAMS, N., JACKSON, H., RALPH, P. & NAKOINZ,

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