/ . Embryol. exp. Morph. Vol. 34, 3, pp. 575-588, 1975
Printed in Great Britain
575
The relationship between erythropoietin-dependent
cellular differentiation and colony-forming
ability in prenatal haemopoietic tissues
By R. J. COLE,1 T. REGAN, 1 S. L. WHITE 1 AND E. M. CHEEK 1
From the School of Biological Sciences, University of Sussex
SUMMARY
Levels of haem synthesis achieved by foetal liver erythroblasts responding to erythropoietin
in vitro are similar in dissociated cell cultures and in cultures of organized tissues. Erythroid
colony-forming cells reach maximum numbers on the sixteenth day of gestation. Their
presence in foetal liver is associated with the period of most rapid production of erythrocytes,
and with in vitro sensitivity to erythropoietin measured as enhanced haem synthesis. It is
concluded that at least a proportion of erythroid colony-forming cells in the foetal liver
are dependent on erythropoietin in situ and that these cells are separated from the earliest
recognizable pro-erythroblast by 1-2 cell divisions. Populations of granulocyte-macrophage
colony-forming cells change independently of erythroid colony-forming cell numbers.
INTRODUCTION
The development of techniques which allow prenatal haemopoietic tissue
to respond to erythropoietin in vitro by enhanced haem synthesis (Cole &
Paul, 1966; Cole, Hunter & Paul, 1968; Bateman & Cole, 1971) or globin
synthesis has facilitated investigation of the molecular events underlying
differentiation of erythroid cells and the initiation of haemoglobin synthesis
(Djaldetti, Preisler, Marks & Rifkind, 1972; Terada et al. 1972; Harrison,
Conkie & Paul, 1973). The cell kinetics of prenatal haemopoietic tissues are
now well documented (Paul, Conkie & Freshney, 1969; Tarbutt & Cole, 1970;
Wheldon et al. 1974). Extension of the spleen-colony assay for haemopoietic
stem cells (Till & McCulloch, 1961) to prenatal mouse haemopoietic organs
has led to detailed descriptions of the changes in numbers of this cell type
during gestation (Silini, Pozzi & Pons, 1967; Moore & Metcalf, 1970). Recent
advances permit the in vitro clonal analysis of progenitor cells of both the
granulocyte/macrophage (Moore & Metcalf, 1970; Moore & Williams, 1973)
and erythroid populations (Stephenson, Axelrad, McLeod & Shreeve, 1971).
Haematological studies on foetuses indicate that during the initial phase of
1
Authors' address: School of Biological Sciences, University of Sussex, Falmer,
Brighton, U.K.
576
R. J. COLE, T. REGAN, S. L. WHITE AND E. M. CHEEK
hepatic erythropoiesis foetal tissues are likely to be relatively poorly oxygenated,
with consequently high levels of erythropoietin or similar erythropoietic
stimulatory factor in the foetal circulation. During later hepatic erythropoiesis,
as a result of a very rapid increase in red cell number, a more stable equilibrium
is reached and erythrocyte production in the liver slows. This is probably a
consequence of both reduced erythropoietin levels, and the operation of
directly acting negative feedback controls on erythroid proliferation or differentiation (Cole, 1975).
The present report is concerned with interrelationships between erythropoietin
sensitivity of mouse foetal liver cells in vitro, when explanted as cell suspensions
and as organized tissues, and the numbers and behaviour of erythroid colonyforming cells (CFUe) and granulocyte-macrophage colony-forming cells
(CFUC) in foetal liver, spleen and bone marrow.
MATERIALS AND METHODS
Animals
Foetal material was obtained from timed natural matings of inbred strain-129
mice. The morning on which mating plugs were found began day 0.
Culture methods
(a) Dissociated cell cultures. Foetal livers were disaggregated to single-cell
suspensions by pipetting in culture medium, without the use of enzymes, and
cultured at 1-2 x 106 cells/ml in Waymouth's medium supplemented with
7-5% foetal calf serum and 2-5% mouse serum. Cultures were maintained at
37 °C and pH 7-4 in 5 % CO2 in air.
(b) Liver fragments. Isolated livers were cut into fragments of approximately
2 mg, excess liquid removed, and their wet weight determined in pre-weighed
tubes containing culture medium. Each fragment was cultured in 3 ml of
medium as above, in sealed 15 ml culture tubes on an inclined roller-tube
apparatus.
(c) Erythroid colony-forming cells. Foetal livers were disaggregated as above.
Foetal spleens were disaggregated by repeated passage through a 0-8 mm
diameter syringe needle, in culture medium. Foetal femurs were dissected free
of adherent tissue, crushed with a glass rod in a small volume of culture medium
and marrow cells suspended by repeated pipetting. Erythroid colony-forming
cells were cultured in plasma clots according to the method of Stephenson
et al. (1971), in medium NCTC 109 supplemented with 10% foetal calf serum,
1% mouse serum, 0-02mg/ml asparagine, 0-85% bovine embryo extract, 1%
bovine serum albumin and 10% citrated bovine plasma. Cultures (1 ml) were
initiated with 1-4 x 105 cells in 35 mm diameter 'Nunc' plastic Petri dishes and
maintained for 3 days in 5% CO2 in air at 37 °C. Colonies were initially
characterized by staining, after fixation with glutaraldehyde and drying, with
Prenatal haemopoietic differentiation
577
Lepehne's stain, or in situ, without drying, with 0-2% benzidine in 0-5 % acetic
acid + 0-4% 30% H2O2. They were routinely counted without staining at
x 100 magnification. Colonies containing more than eight cells and of
characteristic erythroid appearance were counted (Gregory, McCulloch & Till,
1973; Iscove, Sieber & Winterhalter, 1974).
(d) Granulocyte-macrophage colony-forming cells. Cell suspensions were
obtained as above and cultured in 35 mm 'Nunc' plastic Petri dishes in 1-5 ml
modified Eagle's M.E.M. containing 0-3% agar, as described by Metcalf &
Moore (1971). Medium conditioned by mouse L cells grown in suspension was
used as a source of Colony Stimulating Factor (C.S.F.) at 10% (the concentration which elicited maximum numbers of colonies from foetal liver and adult
marrow). Rat-erythrocyte lysate (Bradley, Sumner & Mclnerney, 1974) was
added to this semi-solid agar medium. Cultures were maintained at 37 °C in
5% CO2 in air for 8 days and examined at x 50 magnification for the incidence
of colonies of more than 40 cells. Each experiment with foetal material was run
in parallel with cultures derived from pooled femoral bone marrow of three
or four 25-gm strain-129 male mice. Such cultures invariably gave an incidence
of colonies within the range 2-3 x 103—2-5 x 103/106 marrow cells, so the values
obtained for foetal tissue are presented without normalizing to a notional
bone-marrow standard (Metcalf & Moore, 1971).
BIOCHEMICAL TECHNIQUES
Haem synthesis was estimated by incubating cells or liver fragments in
1-2 ftC\jm\ 59Fe (ferric chloride) previously equilibrated with mouse transferrin
by incubation with 50% mouse serum in culture medium. Haem was extracted
from washed cells with acid ethyl methyl ketone and counted in a gas-flow
counter.
Human urinary erythropoietin lot EPM-3-TaLSL or lot EPM-10-Ta.LSL,
prepared by the Los Angeles Children's Hospital, was used at 0-2 unit/ml for
stimulation of haem synthesis, and at 0-6 unit/ml for estimation of CFU e .
RESULTS
Changes in circulating blood cells and foetal liver
Changes in the peripheral circulation during the hepatic phase of erythropoiesis are shown in Fig. 1. Red-cell concentration rises rapidly until day 16,
then remains relatively constant until near birth. Similar changes are seen for
the concentration of haemoglobin in the blood. Haemoglobin content per unit
of body weight also shows a sharp inflexion on day 16, and falls slightly during
the last 2-3 days of gestation, before initiation of red-cell production by the
spleen and bone marrow. Changes in the rate of accumulation of total erythrocytes/foetus are less sharp, but again there is an inflexion on day 16.
578
R. J. COLE, T. REGAN, S. L. WHITE AND E. M. CHEEK
10
2-8
1
2-4
6
c"
8-]
20
4 cH
60-
o
o
4)
0-8
J? 0-4
13
14
15 16 17 18
Foetal age (days)
19
Fig. 1. Developmental changes in the haematological state of strain 129 foetal
mice. Erythrocyte concentration in the peripheral circulation (O—O) is shown in
relation to haemoglobin content/unit of body weight ( Q — • ) ; haemoglobin content/
unit volume of blood (A—A); and total erythrocytes/foetus ( • — • ) .
100
90 _12
111
13 14
80
Days gestation
15
16
17
I . I
I
18
I
70
I*
|
SO
o
40
A 30
20
10
0
10
20
30
40
Cells/liver x l O " 6
50
60
Fig. 2. Developmental changes in the foetal liver. Total erythroblasts are shown as
a percentage of total liver cells (O—O) together with the changing proportions of
early erythroblasts (pro- and basophilic) (A—A) and late erythroblasts (polyand orthochromatic) ( • — • ) . The arrows indicate the total number of liver cells
characteristic of each day of gestation.
Prenatal haemopoietic differentiation
579
The progression of erythropoiesis in the foetal liver is shown in Fig. 2. The
proportion of erythroblasts in the foetal liver rises rapidly to a maximum on
day 13, is fairly stable until day 15, but then declines very sharply, so that on
day 16 and until birth recognizable erythroblasts represent less than 30% of
the total cellular complement of the foetal liver. A similar progression is seen
in the relative proportions of early and late erythroblasts.
1000
10
12
13
14
15
Foetal age (days)
16
17
Fig. 3. Erythropoietin responsiveness of dissociated foetal liver cells in vitro.
Data expressed/liver complement of erythroblasts for each day of gestation.
Haem synthesis in the first 4 h of culture (O—O) is compared with that during
25-29 h of culture in the presence ( • — • ) and absence (A—A) of erythropoietin.
Changes in the sensitivity of liver erythroblasts to erythropoietin in vitro
The effects of erythropoietin on haem synthesis in cultured foetal erythroblasts were examined in two types of culture system, dissociated cell suspensions
(Fig. 3) and fragments of foetal livers maintained with their tissue structure
intact (Fig. 4). Dissociated foetal liver-cell cultures responded to erythropoietin
by increased haem synthesis up to day 16 of gestation. During the period of
most rapid haem synthesis in the presence of erythropoietin (24-29 h after
explantation) the rate of haem synthesis clearly exceeded that of freshly explanted
37
EHB 34
580
R. J. COLE, T. REGAN, S. L. WHITE AND E. M. CHEEK
10000 i-
1000
100
10
12
13
14
15
Foetal age (days)
Fig. 4. Erythropoietin responsiveness of foetal liver tissue in vitro. Data expressed/
liver for each day of gestation, cultured in the presence (•—Q) and absence
(O—O) of erythropoietin (Ep).
1-6 r
Fig. 5. Changes in the numbers of erythroblasts in dissociated foetal liver cell
cultures. Early ( • — • ) and late ( • — • ) erythroblasts cultured in the presence
of erythropoietin, and early (O—O) and late ( • — # ) erythroblasts without
erythropoietin.
Prenatal haemopoietic differentiation
581
cells and was two to three times that found when erythropoietin was not
present in the culture medium.
The erythropoietin responsiveness of intact foetal liver tissue was comparable
to that observed in dissociated cell cultures prepared from foetuses of similar
gestational age (Fig. 4). Similar levels of haem synthesis per liver were obtained
6r
11
12
13
14
15
16
Foetal age (days)
17
19
Fig. 6. The number (O—O) and percentage ( # — # ) of all erythroid colony-forming
cells in foetal liver, and number (D—D) and percentage ( • — • ) of cells able to
form colonies without exposure to erythropoietin in vitro.
in both types of culture. These results indicate that the maintenance of normaT
cell-cell interactions is not essential for erythropoietin responsiveness or for
normal levels of haem synthesis during short-term culture. Changes in the
relative proportions of early and late erythroblasts in dissociated foetal livercell cultures are shown in Fig. 5. There is some initial increase in the number of
late erythroblasts even in the absence of erythropoietin, but this is only maintained if erythropoietin is present. Early erythroblasts disappear from the
culture unless erythropoietin is present. Such findings are consistent with those
of others, e.g. Harrison et al. 1973, using various strains of mice.
37-2
582
R. J. COLE, T. REGAN, S. L. WHITE AND E. M. CHEEK
Erythroid colony-forming cells in foetal liver
Cells able to form colonies of eight or more haemoglobinized cells after 72 h
culture in semi-solid medium occur in the foetal liver until day 17-18 of gestation (Figs. 6, 7). The maximum number of such cells is found on day 16, when
they represent 1-5-2% of total liver cells. The population doubling time
between days 11 and 16 of gestation is approximately 22 h. Some erythroid
4r
—11-5
12
13
14
15
16
Foetal age (days)
17
18
19
Fig. 7. The absolute number (O—O) and percentage {+—•) of erythroid colonyforming cells dependent on continued exposure to erythropoietin in vitro.
colonies can develop even in the absence of added erythropoietin, but from day
14 to day 16 these were clearly exceeded by erythropoietin-dependent colonyforming cells. Day-17 foetal livers contained only colonies which developed
without added erythropoietin and day-18 foetal livers contained no erythroid
colony-forming cells at all. From day 11 to day 15 of gestation the proportion
of colony-forming cells dependent on additional erythropoietin increased, but
then declined sharply. In contrast, cells able to form colonies independently of
added erythropoietin declined as gestation advanced. The ratio of total erythroid colony-forming cells to erythroblasts (Fig. 8) was 4-5/100 on day 11, and
fell to about half this value until day 15, before reaching a second peak on day
16. The ratio of erythropoietin-dependent colony-forming cells to erythroblasts
was 1/100 until day 13, doubling until day 16, and then declining rapidly.
583
Prenatal haemopoietic differentiation
Erythroid colony-forming cells in foetal spleen and bone marrow
Foetal spleen contains erythroid colony-forming cells by days 16-17 of
gestation (Table 1) and these increase up to birth with a population doubling
time of approximately 20 h, similar to that found in the foetal liver. The
12
13
14 15 16
Foetal age (days)
17
18
19
Fig. 8. The relationship between erythroid colony-forming cells and erythroblasts
in foetal livers; total colony-forming cells (O—O), those independent of erythropoietin in vitro ( • — • ) , and erythropoietin-dependent colony-forming cells
Table 1. Colony-forming cells in prenatal spleen and bone marrow
Erythroid colony-forming cells
Granulocyte-macrophage
colony-forming cells
Spleen
Femur
A
Foetal
age (days)
Spleen
Femur
16-17
17-18
18-19
76 ±10
329 ±52
292 ±23
3
248 ± 20
79 ±15
Total
EP-dependent
Total
EP-dependent
532 ±161
893 ±137
3027 ±449
226 ±117
500
1115 ±510
463 ±82
2129 ±209
1562 ±312
188 ±70
301 ±148
1562 ±142
femoral bone marrow also contains erythroid colony-forming cells by days
16-17 of gestation, and these increase at a similar rate to those in the spleen
in the 2-3 days immediately preceding birth.
Granulocyte-macrophage colony-forming cells in the foetal liver
The number of granulocyte-macrophage colony-forming cells in the foetal
liver reaches a maximum during days 15-16 of gestation and then remains
584
R. J. COLE, T. REGAN, S. L. WHITE AND E. M. CHEEK
fairly stable until birth. The proportion of colony-forming cells in the liver is
maintained at about 0-4% of total liver cells (Fig. 9) but is more variable when
expressed as a proportion of non-erythroid liver cells, reaching a peak (about
2 %) on day 13, but declining in the 4-5 days preceding birth.
Granulocyte-macrophage colony-forming cells in foetal spleen and bone marrow
The foetal spleen already contains a significant number of granulocytemacrophage colony-forming cells by days 16-17 of gestation, and on days 18
li
13
14
15
16
17
18
19
Foetal age (days)
Fig. 9. The number of granulocyte-macrophage colony-forming cells in foetal
liver (O—O) and their percentage of total liver cells ( • — • ) •
and 19 the numbers remain fairly constant, at about 300 CFUc/spleen. Such
cells are rare in day 16-17 femoral bone marrow but increase during days 18-19
of gestation (Table 1).
DISCUSSION
The granulocyte-macrophage colonies which develop in semi-solid media
in the presence of colony-stimulating factor are derived from single cells that
make up a population intermediate between the multi-potent haemopoietic
stem cells, and the histologically recognizable granulocyte precursors. Such
populations, definable in terms of their kinetic properties and their dependence
on specific growth-regulating factors for further differentiation, may conveniently
be termed the 'progenitor cell compartments', and are specific for each line of
haemopoietic differentiation (Metcalf & Moore, 1971). Granulocyte-macrophage progenitor cells, capable of forming colonies in vitro, have been detected
Prenatal haemopoietic differentiation
585
in prenatal mouse yolk sac, liver, spleen and bone marrow (Moore & Metcalf,
1970; Moore & Williams, 1973), although these organs lack marked granulocyte
differentiation in utero. The characteristics of cells which give rise to clonal
erythroid colonies in semi-solid media are less well defined. Transfusion-induced
polycythaemia, accompanied by reduced plasma erythropoietin levels, reduces
the numbers of erythroid colony-forming cells in postnatal spleen and bone
marrow, so that at least some of these erythroid precursors must themselves
be dependent on erythropoietin for differentiation or survival in vivo (Gregory
etal. 1973).
There is no general agreement on the minimum number of cells constituting
an 'in vitro erythroid colony'. In the present study we have adopted the minimum number, eight, used by other workers (Gregory et ah 1973) when undertaking kinetic studies of erythroid colony-forming cells in postnatal haemopoietic tissue. Recent studies (Cooper et ah 1974) using methyl cellulose as a
support medium have shown that both the number of erythroid precursor cells
triggered to form colonies, and the number of haemoglobinized cells within
each colony, are dependent on the concentration of erythropoietin. Using
conditions comparable to those of our present report, 60 %-85 % of colonies
derived from early foetal livers were reported to contain more than eight cells,
and 30%-60% of colonies contained more than 16 cells.
Prenatal haemopoietic tissues appear to contain two types of erythroid
colony-forming cell, one dependent on the continued presence of relatively high
levels of erythropoietin during culture, the other, able to form colonies without
addition of further erythropoietin. The second type may represent relatively
further differentiated cells, already triggered by erythropoietin in the foetal
circulation. Alternatively, there may exist some potential colony-forming cells
that are highly sensitive to erythropoietin and can therefore react to the low
levels present in the serum and plasma in the culture medium. Neither the
absolute nor the relative numbers of erythroid colony-forming cells in developing foetal haemopoietic tissue can be easily correlated with changes in numbers
or proportions of recognizable erythroblasts. In particular, the highest proportion of erythroid colony-forming cells occurs during days 14-15, 2 days after
the peak in early erythroblasts. It is therefore unlikely that the colony-forming
cell is identical to a histologically recognizable early erythroblast.
Kinetic studies of erythropoiesis in prenatal mice (Paul et ah 1969; Tarbutt &
Cole, 1970; Wheldon et ah 1974) suggest that there are four to five cell divisions
in vivo between entry into the pro-erythroblast compartment and the formation
of orthochromatic erythroblasts. If it is assumed that the cloning efficiency of
the erythropoietin-dependent colony-forming cell is fairly high, i.e. not less
than 20 % (as found for the granulocyte-macrophage progenitor by Metcalf &
Moore, 1970) it is likely that this cell type is separated from the earliest recognizable pro-erythroblast by one or two divisions, since colony-forming cells are
present in foetal liver at a level of 1-2/100 erythroblasts.
586
R. J. COLE, T. REGAN, S. L. WHITE AND E. M. CHEEK
The period of rapid rise in absolute numbers of erythroid colony-forming
cells in the foetal livers and their increase relative to liver cells is closely correlated with the period during which dissociated erythroblasts respond to
erythropoietin by increased haem synthesis in vitro, i.e. up to day 15-16 of
gestation. This is also the period during which the amount of haemoglobin,
relative to the mass of the embryo and associated parameters (Fig. 1), is increasing most rapidly. A similar trend is followed by those erythroid colonyforming cells requiring erythropoietin in vitro and by those seemingly independent of further stimulation. The period of relative stability of the parameters
describing the peripheral circulation, e.g. red cell concentration, which begins
on day 16-17 of gestation, is also characterized by loss of erythropoietin
sensitivity in explanted foetal liver cells and by a rapid loss of erythroid colonyforming cells. Erythroid colony-forming cells in the foetal liver therefore only
appear and increase during a period of great demand for, and rapid production
of, erythrocytes, and while the total haemoglobin content of the foetus is low
relative to its mass. This suggests that the maintenance of such colony-forming
cells in the foetal liver is associated with relatively high erythropoietin levels in
the foetal circulation. In prenatal tissue as in postnatal (Gregory et al. 1973),
at least a proportion of the in vitro erythroid colony-forming cells appear
dependent on erythropoietin for their production, as well as their expression.
Initiation of haemopoiesis in foetal spleen and bone marrow follows immigration of precursor cells, probably from the foetal liver, around day 15-16 of
gestation (Petrakis, Pons & Lee, 1969; Metcalf & Moore, 1971). Both types of
erythroid colony-forming cells found in foetal liver are already represented in
day 16-17 spleen and femoral bone marrow. They increase in number, with a
population doubling time similar to that found in foetal liver, over the next
2-3 days, while numbers in the liver decline. Cessation of production of erythroid colony-forming cells by the foetal liver may therefore also involve
changes in the liver microenvironment which render it less able to support
erythropoiesis (Trentin, 1970). Levels of haemoglobin/unit of body weight also
begin to decline on day 17-18, so that the proliferation of erythroid colonyforming cells in both prenatal spleen and bone marrow can be correlated with
an increased demand for erythrocytes.
The progenitor cells forming granulocyte and macrophage colonies in vitro
were found in foetal liver, spleen and bone marrow, together with erythroidcolony progenitor cells. The sharp reduction in the number of erythroid progenitor cells, in later foetal liver, was not matched by any corresponding increase in granulocyte-macrophage progenitors. This underlines the independence
of such haemopoietic cell compartments. The in vitro development of granulocyte-macrophage progenitors from all three prenatal tissues was predominantly
towards macrophage expression. Although this tendency was enhanced by use
of the rat-erythrocyte lysate (Bradley et ah 1974), which also markedly reduced
the proportion of clusters (i.e. colonies with fewer than 40 cells), it is consistent
Prenatal haemopoietic differentiation
587
with the relative frequency of macrophages in prenatal mouse haemopoietic
tissues and the corresponding lack of granulocyte differentiation.
This work was supported by the Medical Research Council.
Erythropoietin was supplied by the Committee on Erythropoietin of the U.S. National
Heart and Lung Institute. It was procured by the Department of Physiology, University of
the Northeast, Corrientes, Argentina, and processed by the Haematology Research
Laboratories, Children's Hospital of Los Angeles.
REFERENCES
A. E. & COLE, R. J. (1971). Stimulation of haem synthesis by erythropoietin in
mouse yolk-sac-stage embryonic cells. /. Embryol. exp. Morph. 26, 475-480.
BRADLEY, T. R., SUMNER, M. A. & MCINERNEY (1974). Observations on enhancement of
mouse marrow cell proliferation by erythrocyte extracts (p. 77). In Haemopoiesius in
Culture (ed. W. A. Robinson), pp. 74-205. Washington, D.H.E.W. publication (NTH).
COLE, R. J. (1975). Regulatory functions of microenvironmental and hormonal factors in
prenatal haemopoietic tissues. In Early Mammalian Development (ed. M. Balls & A.
Wild), pp. 335-358. London: Cambridge University Press.
COLE, R. J., HUNTER, J. & PAUL, J. (1968). Hormonal regulation of prenatal haemoglobin
synthesis by erythropoietin. Br. J. Haemat. 14, 477-488.
COLE, R. J. & PAUL, J. (1966). The effects of erythropoietin on haem synthesis in mouse
yolk-sac and cultured foetal liver cells. /. Embryol exp. Morph. 15, 245-260.
COOPER, M. C, LEVY, J. CANTOR, L. N., MARKS, P. A. & RIFKIND, R. A. (1974). The effect
of erythropoietin on colonial growth of erythroid precursor cells in vitro. Proc. natn.
Acad. Sci., U.S.A. 71, 1677-1680.
DJALDETTI, M., PREISLER, H., MARKS, P. & RIFKIND, R. A. (1972). Erythropoietin effects on
foetal mouse erythroid cells. II. Nucleic acid synthesis and the erythropoietin sensitive cell.
/. biol. Chem. 247, 731-735.
GREGORY, C. J., MCCULLOCH, E. A. & TILL, J. E. (1973). Erythropoietic progenitors capable
of colony formation in culture: state of differentiation. /. cell. Physiol. 81, 411-420.
HARRISON, P. R., CONKIE, D. & PAUL, J. (1973). Role of cell division and nucleic acid
synthesis in erythropoietin-induced maturation of foetal liver cells in vitro. In The Cell
Cycle in Development and Differentiation (ed. M. Balls & F. S. Billett), pp. 341-354.
London: Cambridge University Press.
ISCOVE, N. N., SIEBER, F. & WINTERHALTER, K. (1974). Erythroid colony formation in cultures
of mouse and human bone marrow. Analysis of the requirement for erythropoietin by
gel filtration and affinity chromatography on agarose-concanavalin A. /. cell. Physiol.
83, 309-320.
METCALF, D. & MOORE, M. A. S. (1971). Hemopoietic cells. Amsterdam: North-Holland
Publ. Co.
MOORE, M. A. S. & METCALF, D. (1970). Ontogeny of the haemopoietic system. Yolk sac
origin of in vivo and in vitro colony forming cells in the developing mouse embryo. Br. J.
Haemat. 18, 279-296.
MOORE, M. A. S. & WILLIAMS, N. (1973). Analysis of proliferation and differentiation of
foetal granulocyte-macrophage progenitor cells in haemopoietic tissue. Cell and Tissue
Kinet. 6, 461-476.
PAUL, J., CONKIE, D. & FRESHNEY, R. I. (1969). Erythropoietic cell population changes
during the hepatic phase of erythropoiesis in the foetal mouse. Cell and Tissue Kinet.
2, 283-294.
PETRAKIS, N. L., PONS, S. & LEE, R. E. (1969). An experimental analysis of factors affecting
the localisation of embryonic bone marrow. In Hemic Cells in vitro (ed. P. Fames),
pp. 3-13. Baltimore: Williams & Wilkins.
BATEMAN,
588
R. J. COLE, T. REGAN, S. L. WHITE AND E. M. CHEEK
G., Pozzi, L. V. & PONS, S. (1967). Studies on haemopoietic stem cells in mouse
foetal liver. /. Embryol. exp. Morph. 17, 303-318.
STEPHENSON, J. R., AXELRAD, A. A., MCLEOD, D. L. & SHREEVE, M. M. (1971). Induction of
colonies of haemoglobin synthesising cells by erythropoietin in vitro. Proc. natn. Acad.
ScL, U.S.A. 68, 1542-1546.
TARBUTT, R. G. & COLE, R. J. (1970). Cell population kinetics of erythroid tissue in the
liver of foetal mice. /. Embryol. exp. Morph. 24, 429-446.
SILINI,
TERADA, M., CANTOR, L. N., METAFORA, S., RIFKIND, R. A., BANK, A. & MARKS, P. A.
(1972). Globin mRNA activity in erythroid precursor cells and the effect of erythropoietin.
Proc. natn. Acad. ScL U.S.A. 69, 3575-3579.
TILL, J. E. & MCCULLOCH, E. A. (1961). A direct measurement of the radiation sensitivity
of normal mouse bone marrow cells. Radiation Res. 14, 213-222.
TRENTIN, J. J. (1970). Influence of hematopoietic organ stroma (hematopoietic inductive
micro environments) on stem cell differentiation. In Regulation of Hematopoiesis (ed.
A. S. Gordon), pp. 159-186. New York: Appleton-Century-Crofts.
WHELDON, T. E., KIRK, J., ORR, J. S., PAUL, J. & CONKIE, D. (1974). Kinetics of development of embryonic erythroid cells. Cell and Tissue Kinet. 7, 181-188.
(Received 5 February 1975, revised 29 July 1975)