/ . Embryol. exp. Morph., Vol. 17, 2, pp. 303-318, April 1967
With 1 plate
Printed in Great Britain
303
Studies on the haemopoietic stem cells of
mouse foetal liver
By G. SILINI, L. V. POZZI & S. PONS 1
From the Laboratorio di Radiobiologia Animale, Roma
Cells obtained from the blood-forming organs of donor animals and injected,
under appropriate conditions, into heavily irradiated mice, can colonize, among
other organs, the spleen of the hosts, giving rise to discrete and differentiated
nodules of haemopoietic tissue (Till & McCulloch, 1961). It has been demonstrated that the number of nodules is directly proportional to the number of
injected cells and that such nodules may be regarded as clones arising from single
cells (Till & McCulloch, 1961; Becker, McCulloch & Till, 1963). The postulate
of classical haematological theories (Maximov & Bloom, 1934; Ferrata, 1935)
that a proportion of the cells in blood-forming organs is capable of indefinite
division with renewal of the stem line and of differentiation into mature blood
cells has thus been experimentally demonstrated.
Whether colony-forming cells should actually be regarded as the very primitive stem cells of the haemopoietic tissues (McCulloch, 1963; Lewis & Trobaugh,
1964) or as the earliest step of a differentiative haemopoietic line (Kay & Davies,
1964) has not yet been fully agreed upon. Neither is it clear whether the spleen
colony technique affords a true measure of the total number of stem cells. Therefore it would seem more appropriate, following Till & McCulloch, to retain the
operational terms of * colony-forming cells' (CFC) and of' colony-forming units'
(CFU) for those colony-formers which are found in the spleen of injected hosts.
The technique has so far been applied to a variety of problems concerning
cellular radiobiology (Till & McCulloch, 1961, 1963; McCulloch & Till, 1962,
1964; Duplan & Wolf, 1962; Hanks, 1964; Hellman, 1965; Mekori & Feldman,
1965), experimental haematology (Cudkowicz, Upton, Smith, Gosslee & Hughes,
1964; Lewis & Trobaugh, 1964; McCulloch, Siminovitch & Till, 1964; Duplan,
1963), kinetics of cell populations (Siminovitch, McCulloch & Till, 1963; Till,
McCulloch & Siminovitch, 1964a, b; Siminovitch, Till & McCulloch, 1964),
and immunogenetics (McCulloch & Till, 1963; Till, McCulloch & Siminovitch,
19646).
The object of the present report is to present some observations pertaining to
the field of embryology, made by the use of this technique in connexion with
1
Authors' address: Laboratorio di Radiobiologia Animale, C.S.N. Casaccia del C.N.E.N.,
Roma, Italy.
304
G. SILINI, L. V. POZZI & S. PONS
other radiobiological experiments. Estimates will be given of the number of
CFC in the liver of embryo mice and of the level of haemopoietic activity of
the liver during foetal life and post-natal development.
ANIMALS AND TECHNIQUES
Animals
The experiments were carried out on two strains of mice: a non-inbred Swiss
strain (SW) and a stock of F1 hybrid animals obtained by crossing inbred C3H
males with C57BL female mice. Animals of both sexes were used as hosts, from
the age of approximately 2 to 4 months. In all cases cells were transplanted
between animals of the same strain. Host mice were randomized according to
weight before starting the experiments. Experimental groups consisted of 10-20
animals.
Age of embryos
In a typical experiment, virgin females would be mated overnight from 5.00
p.m. to 8.00 a.m. at which time they would be scored for the presence of vaginal
plug. The age of embryos was determined by taking as zero time the hour at
which vaginal plugs were first seen. Under these conditions, the mice are born
between 18 and 19 days. Because of the interval between mating and appearance
of vaginal plugs, the error introduced in the estimate of embryonic age could be
as much as 15 h.
Cell suspensions
(a) Estimate of the absolute number of nucleated cells. At known regular
intervals after mating, groups of pregnant females were sacrificed by dislocation
of the cervical column, and the foetuses were excised, washed in ice-cold saline
and kept in a moist cold environment. The livers were then dissected free from
other tissues by the use of a dissecting microscope and weighed singly while
still in ice-cold saline. The organs were then immersed in calcium-free Hanks's
fluid, supplemented with 5 % mouse or newborn calf serum, and dissociated by
repeated aspiration into needles of decreasing bore-size. The earliest time at
which convenient amounts of tissue could be obtained was at 12 days after
vaginal plug. From this age, and for the remainder of intra-uterine life, good
monodispersed cell suspensions can be obtained. The number of nucleated cells
in known volumes of the suspension was determined by direct counting in a
Biirker haemocytometer under phase-contrast, after light staining with methylene-blue and lysing of the erythrocytes with diluted acetic acid. Staining of cells
with 0-05 % trypan blue or 0-05 % eosin Y in Sorensen buffer was also employed
in some early experiments to aid in distinguishing between non-viable debris
and possibly viable and therefore unstained cells. This procedure was later
abandoned because of lack of correlation of staining data with the direct
Liver haemopoietic stem cells
305
viability assay by formation of spleen colonies. It should be pointed out that
the reproducibility of the counts between different samples becomes increasingly
unreliable after birth. Probably as a consequence of a relative increase in the
connective tissue of the organ, suspensions of single cells are less easy to obtain.
This may lead to an underestimate of the true cell number.
(b) Estimate of the CFU titre. For these experiments essentially the same procedure was followed on larger amounts of tissue obtained by pooling several
livers. The suspension, made up to about 7 ml with Hanks+mouse serum, was
allowed to stand at 4 °C in a measuring cylinder for 20 min, to obtain sedimentation of the larger fragments by gravity. The upper 5 ml were then collected and
the number of cells counted as above. This cell suspension was adjusted by
dilution to contain the required number of cells to be injected in a 0-2 ml
volume. This volume was injected into the tail vein of the irradiated recipient
mice.
Irradiation of host mice
Whole-body irradiation was given to groups of 36 or fewer animals in a
Perspex box of 40 x 32 cm, divided into cells of 3 x 3 x 10 cm. Radiation was
produced by an X-ray generator operated at 240 kV, 10 mA, 0-5 mm Cu+1-0
mm Al added filtration, HVL 1-6 mm Cu. The dose-rate at the centre of the
container was 24-6 rad/min. The total dose delivered to the mice was 850 rad.
This treatment was found to be adequate, under our experimental conditions,
to reduce the number of endogenous CFU to a level which would result in
fewer than 0-2 spleen nodules per mouse, on an average, with a survival of
70-75 % of the animals at 9-10 days post-irradiation. Non-inbred Swiss mice
showed a larger variation of colony counts, with a few control mice giving as
many as twenty spleen nodules. This resulted in a higher control background
for some experiments and contributed heavily to the larger variability of the
experimental data obtained with these animals.
Spleen-colony counting
Spleens excised from surviving animals 9-10 days after injection were fixed
in Bouin's fluid and the colonies counted by eye.
Histological examinations
Livers from embryos of different ages and from newborn or adult animals
were examined histologically, either as imprint preparations stained with
May-Griinwald and Giemsa or as sliced preparations stained with haematoxylin
and eosin or with May-Griinwald and Giemsa.
306
G. SILINI, L. V. POZZI & S. PONS
RESULTS
Changes in weight and cell number
Changes in the weight and total number of nucleated cells of the liver in Swiss
and hybrid mice at the various ages are shown in Table 1 and in Text-fig. 1.
Within the observation period covered by the experiments the increase in weight
of the organ may be considered linear, and the increase in the number of cells
Table 1. The increase in weight and total cell number of mouse foetal liver
Standard errors of experimental estimates are shown.
Swiss mice
Days after
vaginal
plug
12
13
14
15
16
17
18
19
20
21
Hybrid mice
r
f
Total number of ^
nucleated cells
Liver weight
(g-)
Total number of
nucleated cells
Liver weight
(g.)
00053 ± 00001
00151 ±00004
00349 ±00013
00489 ±00010
00734 ±00019
00785 ±00025
01061 ±00068
00875 ±0-0053
01182 ±00067
—
2-54 ±008x10°
00043 ± 00002
00119 ±00003
00230 ±0-0009
00431 ±0-0025
0-0515 ±00028
00601 ± 0-0023
00959 ±0-0027
00647 ±0-0017
00844 ±00041
00813 ±00038
2-23 ± 0-51
310 ± 0-36
4-67 ± 0-26
5-75 ± 0-41
5-23 ±0-31
5-21 ±0-41
6-46 ±0-77
9-55 ± 0-73
7
xlO
xlO 7
xlO 7
xlO 7
xlO 7
xlO 7
xlO 7
xlO 7
—
2-28 ±019 xlO6
9-23 ± 0 0 8 xlO 6
2-16 ± 0-22 xlO 7
2-76 ± 0-23 xlO 7
3-61 ±0-63 xlO 7
2-76 ±0-33 xlO 7
308 ± 0-34 xlO 7
3-30 ± 0-29 xlO 7
4-58 ± 0-36 xlO 7
4-46±0-21xl0 7
+1
8
12
14
15
16
17
18
19
20
21
Days after vaginal plug
Text-fig. 1. Changes in weight and total cell number of mouse foetal liver. Standard
errors of experimental points are shown. The ordinate for curves A and B appears on
the left-hand and for curves C and D on the right-hand side of the graph. O, SW
strain; A, hybrid strain; —, weight; — , number of cells.
Liver haemopoietic stem cells
307
roughly parallels the weight gain. In absolute terms, the weight and the cell
number of the hybrid mice are lower than those of the Swiss.
Cloning efficiency of cell suspensions
By repeated experiments on cell suspensions obtained from a pool of livers
at each age, it has been possible to follow the cloning ability of liver cells during
pre- and post-natal life. Different numbers of cells from each suspension were
injected at each observation time, and the data given in Table 2 and Text-fig. 2
are average numbers of CFU/105 nucleated cells. They were obtained by averaging the CFU values of each single spleen counted.
Table 2. The average number of CFU/105 nucleated cells
in mouse foetal liver cell suspensions with standard errors
Number of CFU/105 nucleated cells
Days after
vaginal plug
12
13
14
15
16
17
18
19
20
21
23
26
28
40
Adult
( Swiss mice
2-94 ±0-44
2-35 ±0-74
1-60 ±0-20
2-74 ±0-55
2-74 ±0-37
3-62 ±0-64
4-24 ±0-44
3-85 ±0-43
3-64±l-41
3-47 ±0-64
—
—
1-72 ±0-97
0-83 ±0-24
2-44 ±0-73
A
Hybrid mice
13-25 ±0-84
9-42 ±0-44
9-47 ±0-67
7-58 ±0-31
7-35 ±0-54
7-26 ±0-55
9-48 ±0-44
8-96 ±0-40
—
—
3-87 ±0-29
1-60 + 0-24
—
0-27 ±012
004 ±001
In the Swiss strain the CFU value at 12 days is around 3 per 105 nucleated
cells. After a decrease at 13 and 14 days the CFU value increases gradually to
reach a maximum of 4-3 at 18 days, just before birth. The difference between the
points at 14 and 18 days is statistically significant and the regularity of the upward trend during this time would tend to confirm the increase as real. After
18 days a decrease takes place, reaching a minimum of less than 1 CFU per 105
nucleated cells at 3-4 weeks after birth. Judging by this test, the haemopoietic
function of the liver never ceases entirely in this strain and even at about 2
months of age some colony-forming cells are found.
In hybrid mice the cloning efficiency of the cell suspensions is considerably
higher than in Swiss. The data do not allow the identification of a definite trend
before birth, but from then on a progressive decrease of the cloning efficiency
of the cells takes place to CFU values of virtually zero in the adult animal. This
308
G. SILINI, L. V. POZZI & S. PONS
observation is consistent with a complete loss of haemopoietic capacity of the
liver in the adult.
Cytological analysis
Imprint preparations of the liver-cell population at the various ages were
scored microscopically for differential counting of the various cell types, in view
of a possible correlation between these counts and the data on cloning efficiency.
Counting was done on at least 1000 haemopoietic cells at each age, by the same
observer, with occasional checks by a second one on some of the points. Owing
30
35
40
60
Days after vaginal plug
Text-fig. 2. Changes in the average number of CFU per 105 nucleated liver cells at
various ages. Standard errors of experimental points are shown. The numbers after
each point are the numbers of spleens counted in different titrations at each
observation time. O, SW strain; #, hybrid strain.
to the difficulty in identifying some classes of haemopoietic cells in the mouse
(Dunn, 1954), a detailed classification would hardly be meaningful. It was
therefore decided to group the cells as follows: (a) hepatocytes and other cells:
under the conditions of fixation and staining used, they can be recognized on the
basis of their very large size and a pink cytoplasm with a very fine reticular
structure; (b) pronormoblasts: cells with a large round purple heavily stained
nucleus with fine chromatin structure and with basophilic granular cytoplasm;
nucleoli are present; (c) normoblasts: small cells with a compact and darkstaining nucleus and with polychromatophylic or acidophylic cytoplasm; (d)
myeloblasts: large cells with a big round nucleus containing coarse chromatin
No.
771
1816
1078
803
530
983
1520
1869
1928
2764
2660
6948
9912
Days
after
vaginal
plug
12
13
14
15
16
17
18
19
20
21
22
24
Adult
648
610
675
543
551
588
265
158
341
198
213
86
0
No.
*
No.
0/ *
/o
Normoblasts
No.
/o
0/
*
Myeloblasts
No.
/o
0/
*
Myelocytes
No.
/o
0/
*
Metamyelocytes
6-3
282
28-2
5
2
63
0-5
0-2
64-8
31
201
131
201
27
131
2-7
31
610
6-7
22-7
227
15
16
67-5
1-5
1-6
67
14-8
69
54-3
6-9
201
40
201
40
148
72
551
24-5
101
7-2
31
245
31
101
6-5
123
163
12-3
61
61
58-8
16-3
65
292
71
27-3
29-2
273
26-5
100
100
71
80
390
25-8
258
390
80
11-4
15-8
114
221
341
22-1
40
361
3-6
361
36
40
5-2
414
24-9
41-4
249
88
52
19-8
8-8
250
4-6
250
21-3
42-9
58
429
46
5-8
5-6
578
13-9
41
8-6
139
57-8
41
56
11
15-9
00
12-5
63
00
71-5
0
14
* Percentag*j of haemopoietic cells (nucleated erythrocytes excluded).
t Percentage of total nucleated cells (nucleated erythrocytes excluded).
/o
0/
Hepatocytes and
other Pronormoblasts
Table 3. Differential counting of cell types in the liver of hybrid mice
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
88
No.
A
56-4
35-5
481
55-4
65-3
50-4
39-6
34-8
341
26-5
27-3
12-5
0-9
%t
Total
haemopoietic
cells
1771
2816
2078
1803
1530
1983
2520
2869
2928
3764
3660
7948
10000
counted
CCJJ-O
Total
O
«>'
a
310
G. SILINI, L. V. POZZI & S. PONS
granules and a small amount of basophylic non-granular cytoplasm; (e)
myelocytes: cells with a typically bean-shaped nucleus; (/) metamyelocytes:
cells with a doughnut-shaped nucleus which shows a fine pink chromatin
structure.
Imprint preparations of liver cells obtained from animals at two different
foetal ages are shown in Plate 1 and some cell types can easily be recognized.
Table 3 and Text-fig. 3 show the relative changes of these cell types in the
hybrid mouse strain with increasing foetal age of the animals. The proportion
60
50
•
30
"3
3 20
g
10
10 11
12 13 14
15 16
17 18
19
20 21 22 23 24
60
Days after vaginal plug
Text-fig. 3. Data on differential counting of haemopoietic cell types in the foetal
liver of hybrid mice. • , Pronormoblasts; O, normoblasts; m, myeloblasts; • ,
metamyelocytes; A, myelocytes.
of haemopoietic cells over the whole liver-cell population is around 50 % during
foetal life and decreases gradually during the first week after birth and throughout post-natal life to a level of practically zero in the adult animal. The percentage of myelocytes and myeloblasts remains unchanged up to 24 days after
vaginal plug, whilst the more differentiated myeloid cells show an increase
from the earliest times of observation. Metamyelocytes amount to about 50 %
of all haemopoietic cells a week after birth. The percentage of pronormoblasts
falls after about 17 days and this fall is followed by a drop in the normoblast
counts which begins about 3 days later.
/. Embryol. exp. Morph., Vol. 17, Part 2
PLATE 1
t
Imprint preparations of foetal liver cells stained with May-Griinwald and Giemsa. Fig. A,
12-day-old liver, x 320. Fig. B, 12-day-old liver, x810. Fig. C, 17-day-old liver, x 320.
Fig. D, 17-day-old liver, x810. A few cell types are shown as follows: n. = normoblast;
h. = hepatocyte; n.e. = nucleated erythrocyte; m. = metamyelocyte. Note the relative
increase in metamyelocytes in C and D.
G. SIL1NI, L. V. P O Z Z I & S. P O N S
facing p. 310
Liver haemopoietic stem cells
311
Estimate of the number of CFU per liver
In principle, it should be possible to calculate the absolute number of CFU
in the foetal liver at a given age by a simple multiplication of the absolute
number of cells and the cloning efficiency of these cells in the spleen of the host
mice. The results of such calculations are given in Table 4. It should be realized
that the error affecting these estimates is likely to be fairly large, since it reflects
the variability in both the cell counts and in the estimate of the cloning efficiency.
In addition, owing to the fact that the absolute number of nucleated cells in the
Table 4. The total number of CFU in the foetal liver of Swiss and Hybrid mice
The errors shown are based on the variability of total cell counts.
Total CFU/liver
Days after
vaginal plug
12
13
14
15
16
17
18
19
20
A
f
Swiss mice
Hybrid mice
75 ± 3
524 ±120
496± 57
1280± 71
1575±115
1893 ±112
2209 ±174
2487 ± 295
3476 ±264
302± 25
867± 95
2045 ± 208
2092+167
2653 ±463
2003 ± 239
2920 ±322
2956 ± 260
—
Table 5. The number of CFUIliver in hybrid embryo mice of various ages
StandarcI errors of experimental points are shown.
Number of CFU/liver
Days after
vaginal plug
12
15
17
19
Indirect estimate
2
302 ± 0-25 xlO
20-90 ± 1 -70 xlO2
2000 ± 2-30 xlO2
29-50 ± 2-60 xlO2
A
Direct estimate
5-15 ± 0-14 xlO2
3615 ± 9-20 xlO2
54-47 ± 108 xlO2
34-06 ± 1-18 xlO2
liver is probably underestimated by the technique used, it should be expected
that the calculated absolute numbers of CFU may be lower than the real value.
The data show that the number of liver stem-cells increases considerably between
day 12 and day 14. From then on there is a slowing down of the rate of increase
until the time of birth. Up to the time of birth there is, however, no sign of
decline in the haemopoietic activity of the liver, which continues irrespective
of the marrow haemopoiesis that has been established in the meantime, 3-4 days
before term (Barnes, Ford & Loutit, 1964). It would be expected that after birth
the number of stem-cells should fall rapidly in a way that roughly parallels the
fall of the CFU value.
20
JEEM 17
312
G. SILINI, L. V. POZZI & S. PONS
These indirect estimates of absolute values of CFU were checked by direct
experimental determinations obtained by the injection of cell suspensions from
a single liver, after appropriate dilution. These are very demanding experiments
in terms of the number of host animals and they have therefore been carried
out on hybrid animals at only four different times. The results are given in
Table 5 and show that throughout foetal life the estimate of the CFU content of
the liver obtained by direct determination is higher by a factor of about two as
compared with the calculated value. For the reasons given above, these latter
estimates should be considered as more truly representative of the absolute
content of CFU of the liver.
Estimate of the absolute number of CFC per liver
The data reported in the preceding section provide an estimate of the number
of liver cells which are capable of colonizing the spleen of appropriately conditioned hosts. It is known (Siminovitch et al. 1963), however, that only a
Table 6. The proportion of injected CFC from foetal liver capable
of colonizing the spleen of host mice
Standard errors of experimental points are shown.
/factor (%)
Days after
vaginal plug
12
15
17
18
19
A
r
Swiss mice
—
—
9-41 ±0-55
—
—
Hybrid mice
4-99 ±0-57
10-60 ±0-58
8-50 ±0-65
8-79 ±0-67
12-69 ±0-63
proportion of all the potential colony formers that are injected may settle in the
spleen and it is conceivable that this fraction may be related to at least two factors : the blood supply of the spleen and its ability to provide suitable growth
conditions to the injected stem cells. The knowledge of this fraction/, which may
be regarded as a measure of the cloning efficiency of the colony-formers in the
spleen, is essential for an absolute estimate of the total number of CFC.
A technique for the estimation of the /factor has been described previously
(Siminovitch, McCulloch & Till, 1963). In essence, it is based on re-transplanting
into new, conditioned hosts all the cells contained in the spleen of an irradiated
intermediate mouse injected with a suspension of haemopoietic cells of known
CFU titre. The ratio of the colonies arising from the stem-cells contained in the
spleen of the intermediate host to the number of colonies to be expected from
the total CFU injected into the same animal provides an estimate of/.
This experimental scheme was carried out under our experimental conditions
Liver haemopoietic stem cells
313
and the results are given in Table 6. The data show an increase in the percentage
of cells capable of colonizing the spleen between day 12 and day 15. At later
times there is no definite trend and the/factor tends to level off. The conclusion
is drawn that the CFU cannot be regarded as a fixed percentage of the CFC
at all stages of foetal life.
With the knowledge of this fraction/it becomes possible to calculate in the
hybrid mouse strain the absolute number of colony-forming cells which are
present in the liver at the various foetal ages. The results appear in Table 7 and
show a large expansion of the pool of CFC up to the time of birth.
Table 7. The absolute number of CFC in the foetal liver
of hybrid mice at various ages
Days after
vaginal plug
Total no. of
CFU/liver
/factor
(%)
Absolute no. of
CFC/liver
12
15
17
19
302
2092
2003
2956
4-99
10-60
8-50
12-69
6052
19735
23564
23293
DISCUSSION
Some aspects of this research might have been anticipated from a knowledge
of basic descriptive haematology in the foetal and newborn mammal. The changes
in the activity of the liver as a blood-forming organ, such as the progressive
decrease in the percentage of haemopoietic cells after birth paralleled by a drop
n the number of CFC, were to be expected. But the exact timing of these changes
and their quantitative assessment represent, to our knowledge, new findings
worthy of some comment.
Estimates of weight and of the number of cells during the growth of the liver
in foetal life and in early postpartum days have already been reported (Williamson, 1948; Dick, 1956; Widdowson & McChance, 1960; Enesco & Leblond,
1962; Oliver, Ballard, Shield & Bentley, 1962). These studies were carried out
mainly in the rat, in connexion with a theory of liver growth under the regulation
of plasma protein concentration, the evidence for which has been summarized
by Glinos (1958). The theory was founded on experimental data obtained in the
adult liver after hepatectomy and little attention was paid to the fact that the
morphological and functional aspects of the foetal liver are entirely different
from those in the adult animal. It is conceivable that the mechanisms controlling
the growth of the organ are entirely different in the two cases. The progressive
disappearance of the haemopoietic cells and the increase in hepatocytes which
takes place from the time of birth may well explain the decrease in the rate of
growth of the liver during the 3 weeks after birth (Oliver et al. 1962). Under our
experimental conditions, the weight gain of the foetal liver between days 12 and
314
G. SILINI, L. V. POZZI & S. PONS
16 is directly related to and almost entirely accounted for (within the technical
error) by the increase in the number of cells; the slower rate of increase of cell
number compared to the weight gain observed from day 17 cannot be attributed
with certainty to any specific cause, owing to the low resolution of our counting
technique.
CFU values of liver cell suspensions which are low in comparison with the
CFU of adult bone marrow cells have already been reported (Duplan, 1963;
McCulloch & Till, 1963). These observations should be attributed to (a) the
presence in the liver cell suspensions of a large proportion of non-haemopoietic
cells (about 50 %, according to Table 3); (b) the lower / value of liver colonyformers, in comparison with the/reported for bone-marrow cells—17 % in the
experiments of Siminovitch et al. (1963) against a maximum of about 12% in
our experiments (see Table 6). In accordance with point (a), the average CFU
value of the hybrid liver, which is about 8 per 105 cells in our experiments,
would actually be of the order of 16 per 105 cells if the hepatocytes were discarded and would be further increased to about 22 per 105 cells on account of
point (b). This value compares very favourably with a CFU of 2-4 per 104 cells
obtained in our hybrid strain upon transplantation of adult bone marrow cells
(Silini & Maruyama, 1965), The conclusion therefore seems justified that colonyformers amount to about the same percentage of the total haemopoietic cells
both in the foetal liver and in adult bone marrow.
The present experiments have shown changes in the cloning efficiency of
liver cell suspensions with the age of the donor foetuses in the spleen of host
mice. It should be pointed out that the technique for the estimate of this factor
is independent of any counting error of the cell suspension used. The only way
in which technical faults may have influenced the experimental results is through
differential killing of the stem cells obtained from younger livers, during the
resuspension of the spleen of intermediate hosts. In view of the fact that CF
cells represent a very low percentage of all blood-forming cells and because of
the difficulties in the identification of these cells in a cell suspension, any
experimental test of differential killing is at the moment impossible. Our experimental observation seems however of immediate relevance to those radiobiological experiments where the repopulating ability of foetal haemopoietic
cells obtained from foetuses of different ages is compared (Duplan & Wolf,
1962).
McCulloch & Till (1963) have reported that transplantation of haemopoietic
cells between genetically different mouse strains gives a lower yield of spleen
nodules than in isologous transplants. Apparently the heavy radiation treatment
given to the hosts in the course of the CFU titration allows a transplant to
remain viable even in a non-genetically homogeneous system, but it is not
sufficient to inhibit completely the immune reaction against the foreign transplanted tissue. It seems reasonable to assume, on the basis of these results, that
the CFU value of the non-inbred Swiss mouse, generally lower by a factor
Liver haemopoietic stem cells
315
of 2 or 3 with respect to the CFU of the hybrid strain, may be due to a residual
homotransplant reaction.
No clear-cut explanation can be offered for the observation that colonyforming cells in the liver of adult Swiss mice never completely disappear. The
sort of evidence for the presence of foci of erythopoietic cells seen in other
strains (J. F. Duplan, personal communication) has not been found in histological or cytological preparations of adult Swiss livers. This observation cannot
be accounted for by the higher variability of the control, already mentioned, since
the values given in Text-fig. 2 have the background control values subtracted. It
is, however, possible that the nodules observed may be of endogenous origin,
elicited in a non-genetically compatible system as a non-specific reaction to the
large number of cells injected because of the low CFU value of the adult cell
suspensions (Pons & Petrakis, 1964; Smith, 1964).
Evidence has recently been reported that the small lymphocytes are to be
considered the stem-cells of the adult bone marrow and of the circulating blood
n the mouse (Cudkowicz, Bennett & Shearer, 1964). Such experiments have
not been extended to haemopoietic cells of foetal origin and the identification
of the stem cells in the liver remains an entirely open problem. It is not inconceivable that such cells may have a different cytological aspect during foetal life;
in any case the identification on the basis of purely morphological criteria is
made uncertain because of the low content of CFC in the liver. In this respect,
the analysis of the cell population carried out in the present series of experiments,
apart from providing useful quantitative data not available up to now, is of
little help. Experiments presently under way on the relative abundance of
different types of nodules in the spleen of animals injected with these same cell
suspensions may give additional information (Silini & Pons, unpublished data).
The present experiments have shown a quite consistent increase in the absolute
number of CFC in the foetal liver, by about a factor of four between day 12 and
day 17, followed by a tendency towards decrease. This finding raises some
problems regarding the kinetics of the stem cells in a tissue which, unlike the
adult bone marrow, shows an increasing stem cell content. It seems obvious that
this condition should require the presence of regulatory mechanisms at the
cellular and/or the population level, different from those operating in adult
haemopoietic tissues. Data on the regulation of foetal haemopoiesis are to this
date rather scanty. Jacobson, Marks & Gaston (1959) have shown that hypertransfusion of pregnant animals results in a profound depression of maternal
erythropoiesis while haematological values of the foetuses are higher than
normal. Stohlman et al. (1964) have studied the effect of nephrectomy and
starvation in neonatal rodents and have suggested that in the newborn animals
erythropoiesis is governed by factors other than those seen in the adult. At the
cellular level Cole & Paul (1966) have found that liver cells from 11 to 15day foetuses respond to erythropoietin by an increased rate of haem synthesis
while cells from older foetuses seem to be unresponsive. This is evidence for a
316
G. SILINI, L. V. POZZI & S. PONS
differential control of haemopoiesis in foetal and adult tissues to which the
present experiments have added data at the population level.
Further work is required to test whether the models of erythropoiesis developed for adult bone marrow (Lajtha & Oliver, 1960; Lajtha, 1962) and the
theoretical analysis of the increase of stem cells in transplanted spleen nodules
(Till et al. 1964a) also apply to the foetal liver system. It also seems desirable to
obtain additional quantitative information about the functional state of the
stem cells of foetal origin as compared to those in the bone marrow.
SUMMARY
Changes in the stem-cell pool of the mouse foetal liver at various times postconception have been followed using the technique of spleen nodules formation.
A fourfold increase in the absolute number of colony-forming cells has been
found during the 7 days preceding birth. In addition, changes in the transplantability of liver-cell suspensions with foetal age have been revealed. These
data are discussed in relation to the regulatory mechanisms of foetal haemopoiesis.
RIASSUNTO
Studi sulle cellule staminali emopoietiche delfegato embrionale del topo
Usando la tecnica delle colonie spleniche, si sono seguite le variazioni
numeriche di cellule staminali del fegato embrionale di topo a varie eta fetali.
Nei sette giorni precedenti la nascita si e riscontrato un aumento di un fattore
quattro nei numero assoluto di cellule capaci di formare colonie. Si sono
inoltre dimostrate variazioni nella trapiantabilita di sospensioni cellulari di
fegato alle varie eta del feto. Questi dati vengono discussi in relazione ai
meccanismi di regolazione della emopoiesi fetale.
The skilled technical assistance of Mr B. Bassani is gratefully acknowledged. We are
grateful to Professor E. Borghese, Istituto di Anatomia Umana Normale, Universita di
Napoli, and to Dr L. G. Lajtha, Paterson Laboratories, Christie Hospital and Holt Radium
Institute, Manchester, for stimulating discussion.
REFERENCES
D. W. H., FORD, C. E. & LOUTIT, J. F. (1964). Haemopoietic stem cells. Lancet i,
1395-6.
BECKER, A. 1.,-MCCULLOCH, E. A. & TILL, J. E. (1963). Cytological demonstration of the
clonal nature of spleen colonies derived from transplanted mouse marrow cells. Nature,
Lond. 193, 452-4.
COLE, R. J. & PAUL, J. (1966). The effect of erythropoietin on haem synthesis in mouse yolk
sac and cultured foetal liver cells. /. Embryol. exp. Morph. 15, 245-60.
CUDKOWICZ, G., BENNETT, M. & SHEARER, G. M. (1964). Pluripotent stem-cell function of the
mouse marrow 'lymphocyte'. Science, N.Y. 144, 866-8.
CUDKOWICZ, G., UPTON, A. C , SMITH, L. H., GOSSLEE, D. G. & HUGHES, W. L. (1964).
An approach to the characterization of stem cells in mouse bone-marrow. Ann. N. Y. Acad.
Sci. 114, 571-82.
BARNES,
Liver haemopoietic stem cells
317
DICK, D. A. T. (1956). Growth and function in the foetal liver. /. Embryol. exp. Morph. 4,
97-109.
DUNN, T. B. (1954). Normal and pathologic anatomy of the reticular tissue in laboratory
mice, with a classification and discussion of neoplasms. /. natn. Cancer Inst. 14,1281-1390.
DUPLAN, J. F. (1963). Etude sur la structure et la transplantabilite des nodules spleniques
provoquees par l'injection de cellules hemopoietiques foetales a des souris isologues
irradiees. C. r. Seanc. Soc. Biol. 157, 286-9.
DUPLAN, J. F. & WOLF, N. S. (1962). Age-related factors which influence the value of the
mouse embryo for post-irradiation restoration of the adult. Int. J. Radiat. Biol. 5, 597-607.
ENESCO, M. & LEBLOND, C. P. (1962). Increase in cell number as a factor in the growth of
the organs and tissues of the young male rat. /. Embryol. exp. Morph. 10, 530-62.
FERRATA, A. (1935). Le Emopatie. S.E.L. Milano.
GLINOS, A. (1958). The mechanism of the liver growth and regeneration. In The Chemical
Basis of Development (ed. McElroy and Glass), pp. 813-19. Baltimore: Johns Hopkins
Press.
HANKS, G. A. (1964). In vivo migration of colony-forming units from shielded bone-marrow
in the irradiated mouse. Nature, Lond. 203, 1393-5.
HELLMAN, S. (1965). Circulating stem-cells: variation with duration of partial-body Xirradiation. Nature, Lond. 205, 100-1.
JACOBSON, L. O., MARKS, E. K. & GASTON, E. O. (1959). Studies on erythropoiesis. XII. The
effect of transfusion-induced polycytemia in the mother on the foetus. Blood 14, 644-53.
KAY, H. E. M. & DA VIES, A. J. S. (1964). Histological aspects of haemopoietic regeneration.
Brit. J. Radiol. 37, 802 only.
LAJTHA, L. G. (1962). Stem-cell kinetics and erythropoietin. In Erythropoiesis, ed. Jacobson
and Doyle. New York: Grune and Stratton.
LAJTHA, L. G. & OLIVER, R. (1960). Studies on the kinetics of erythropoiesis: a model of the
erythron. In Ciba Fdn Symp. on Haemopoiesis, ed. Wolstenholme and O'Connor. London:
Churchill.
LEWIS, J. P. & TROBAUGH, F. E. (1964). Haematopoietic stem-cells. Nature, Lond. 204, 58990.
MAXIMOV, A. & BLOOM, W. (1934). Textbook of Histology. Philadelphia: Saunders.
MCCULLOCH, E. A. (1963). Les clones de cellules hematopoietiques in vivo. Rev.fr. Etud. din.
biol. 8, 15-19.
MCCULLOCH, E. A., SIMTNOVITCH, L. & TILL, J. E. (1964). Spleen-colony formation in anemic
mice of genotype WWV. Science, N. Y. 144, 844-6.
E. A. & TILL, J. E. (1962). The sensitivity of cells from normal mouse bonemarrow to gamma radiation in vitro and in vivo. Radiat. Res. 16, 822-32.
MCCULLOCH, E. A. & TILL, J. E. (1963). Repression of colony-forming ability of C57BL
haemopoietic cells transplanted into non-isologous hosts. /. cell. comp. Physiol. 61,
301-8.
MCCULLOCH, E. A. & TILL, J. E. (1964). Proliferation of haemopoietic colony-forming cells
transplanted into irradiated mice. Radiat. Res. 22, 383-97.
MEKORI, T. & FELDMAN, M. (1965). Protection of X-irradiated mice by 'cloned' haemopoietic
cells. Transplantation 3, 98-113.
OLIVER, I. T., BALLARD, F. J., SHIELD, J. & BENTLEY, P. J. (1962). Liver growth in the early
post-partum rat. Devi Biol. 4, 108-16.
PONS, S. & PETRAKIS, N. L. (1964). Erythropoietic colony formation in spleen of supralethally irradiated C3H mice following.injection of human lymphocytes and bone-marrow.
Exp. Haematol. 7, 84 only.
SILINI, G. & MARUYAMA, Y. (1965). X-ray and fast neutron survival response of 5-bromodeoxycytidine-treated bone-marrow cells. Int. J. Radiat. Biol. 9, 605-10.
MCCULLOCH,
SIMINOVITCH, L., MCCULLOCH, E. A. & TILL, J. E. (1963). The distribution of colony-
forming cells among spleen colonies. / . cell. comp. Physiol. 62, 327-36.
SIMINOVITCH, L., TILL, J. E. & MCCULLOCH, E. A. (1964). Decline in colony-forming ability
of marrow cells subjected to serial transplantation into irradiated mice. / . cell. comp.
Physiol. 64, 23-31.
318
G. SILINI, L. V. POZZI &S. PONS
W. W. (1964). Haemopoietic colony formation in irradiated mice treated with endotoxin. Exp. Haematol. 7, 80-1.
STOHLMAN, F., LUCARELLI, G., HOWARD, D., MORSE, B., LEVENTHAL, B. (1964). Regulation
of erythropoiesis. XVI. Cytokinetic patterns in disorders of erythropoiesis. Medicine 43,
651-60.
TILL, J. E. & MCCULLOCH, E. A. (1961). A direct measurement of the radiation sensitivity of
normal bone-marrow cells. Radiat. Res. 14, 213-22.
TILL, J. E. & MCCULLOCH, E. A. (1963). Early repair processes in marrow cells irradiated
and proliferating in vivo. Radiat. Res. 18, 96-105.
TILL, J. E., MCCULLOCH, E. A. & SIMINOVITCH, L. (1964a). A stochastic model of stem-cell
proliferation based on the growth of spleen colony-forming cells. Proc. natn. Acad. Sci.,
U.S.A. 51, 29-36.
TILL, J. E., MCCULLOCH, E. A. & SIMINOVITCH, L. (19646). Isolation of variant cell lines
during serial transplantation of haemopoietic cells derived from foetal liver. J. natn. Cancer
Inst. 33, 707-15.
WEDDOWSON, E. M. & MCCHANCE, R. A. (1960). Some effects of accelerating growth. I.
General somatic development. Proc. R. Soc. B, 152, 188-92.
WILLIAMSON, M. B. (1948). Growth of liver in foetal rats. Growth, 12, 145-7.
SMITH,
{Manuscript received 23 September 1966, revised 19 December 1966)