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MICROCOPY RESOLUTION TEST CHART
NATIONAL BUREAU OF STANDARDS -1963
a
Orinn -p-
MASTOD
Cort-660423-/
Proceedings of the International Symposium
"The Lymphocyte in Immunology and Haemo-
poiesis" Bristol, England, April 12-16, 1966
JUN 27 1966
CESTI PSICAS
H.C. $2.00; Mn.50
FUNCTIONAL AND MORPHOLOGICAL CHARACTERIZATION OF
STEM CELLS: THE UNIPOTENTIAL ROLE OF
"LYMPHOCYTES" OF MOUSE MARROW
Michael Bennett and Gustavo Cudkowicz
Department of Experimental Biology
Roswell Park Memorial Institute, Buffalo, New York and
Biology Division, Oak Ridge National Laboratory
Oak Ridge, Tennessee, U. S. A.

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RELEASED FOR ANNOUNCEMENT
LEGAL NOTICE
This repor; was propared as an account of Govornment sponsored work. Neither the United
Statos, nor the Coumission, nor any person acting on behalf of the Commission:
A. Makes anyurranty or representation, expressed or impliod, with respect to the secu-
racy, completoncos, or usefulness of the information contaiaod in the report, or that the uso
of any information, printus, method, or proceso disclosed in this report may not Infringe
privately owned righw; or
B. Assumes any lizbilities with respect to the use of, or for damagos resulting from the
uso of any information, apparatus, mothod, or procoos disclosod in thio report.
Ao used in the above, person acting on behalf of the Commission" Inolude, any on-
ployee or contractor of the Commission, or employee of such contractor, to the oxtent that i
such employee or contractor of the Commission, or employee of such contractor preparos,
disseminates, or provides access to, any information pursuant to his omployment or contract
with the Commission, or is omploymon' with such contrastor.
IN NUCLEAR SCIENCE ABSTRACTS
* Research supported in part by the U.S. Atomic Energy Commission under
contract with Union Carbide Corporation.
.
-
Running title:
Transplantation of Marrow "Lymphocytes''
Send proofs to:
Dr. Gustavo Cudkowicz
Department of Experimental Biology
Roswell Park Memorial Institute
666 Elm Street
Buffalo, New York 14203 U.S. A.
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Introduction
The bone marrow and other organs of the hematopoietic system of
mice consist of a population of cells heterogeneous with respect to the pat-
discussion, any hematopoietic cell population can be subdivided into (a)
differentiating and dividing cells recognizable by morphological or functional
criteria as belonging to the erythroid, myeloid, lymphoid or other series,
and (b) undifferentiated dividing precursor cells not recognizable as belong-
ing to any of the differentiating series. The latter are designated as stem
cells: by definition, stem cells are self-maintaining and capable oi regulated
production of differentiating descendants.
Morphologic characterization of stem cells is difficult because of
the lack of clear-cut features aggravated by the complex heterogeneity of
hematopoietic tissues. However, recent advances in the field of experimen-
tal cellular hematology have brought under investigation the functional char-
acteristics of stem cells. For example, one convenient method of studying
primitive hematopoietic cells is the transplantation of marrow into histo-
compatible host animals exposed to lethal or near-lethal doses of X rays.
Success of the graft depends on proliferation and differentiation of stem
cells seeding the host's hematopoietic organs. Thus, quantitative and quali-
tative definition of the various classes of celle produced by grafted marrow
may characterize the primitive precursors. Furthermore, stem cell
functions assessed in this way may be correlated with the presence and fre-
1..
. -
WAT
T
quency of given recognizable cell types in the donor marrow, providing
thereby indirect evidence for the morphological identification of stem cells.
Methods of investigating primitive marrow cells have been devel-
oped in our laboratory (1, 2) and elsewhere (3, 4). Till and McCulloch
(5) first demonstrated that marrow stem cells may be assayed by the num-
ber of discrete nodular colonies appearing in the spleen of lethally irradi.
ated mice ten days after irradiation and transplantation. On the assump-
tion that each nodule represents a clone (6), this method enumerates the
primitive colony-forming cells lodging in the recipient spleene; but it does
not measure the product of these cells, namely, the expanding hematopoi.
etic population. The latter information is obtained by assessing the pool
size of donor-derived cells engaged in the synthesis of DNA, which in-
cludes undifferentiated and differentiating cells as well as erythroid and
non-erythroid elements. The latter two types can be estimated separately,
. .. .'.
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either by assessing the size of the cell population engaged in synthesis of
.
1
F.
hemoglobin, or by assessing the pool size of cells engaged in synthesis of
..
"
DNA in plethoric recipients, in which recognizable erythropoiesis has been
suppressed experimentally. Using radioactive precursors to label cells
synthesizing DNA and hemoglobin it is possible to quantitate hematopoietic
cell production in vivo and to follow patterns of proliferation and differen-
tiation.
The present report describes experiments which indicate that eryth-
ropoiesis and production of non-erythroid cells in radiation-induced marrow
M
.
5
.
chimeras are independent processes, at least during the early phases of
hematopoietic reconstitution. Presumably, this reflects the existence in
marrow of independent precursor cells recognizable by functional criteria.
This conclusion is based upon evidence interpreted by us as indicating that
the rate of proliferation and production of non-erythroid cells (i. e., cells
not engaged in synthesis of hemoglobin) is not affected by induced changes
in the rate of proliferation of erythroid cells. The experiments also dis-
close a consistent positive correlation between the ability of marrow to pro-
duce erythroid cells on transplantation and its content of small and medium-
sized "lymphocytes." Marrow "lymphocytes" are indistinguishable mor-
phologically from peripheral lymphocytes in the mouse, but they differ with
respect to functional properties. They are neither immunologically compe-
tent (7) nor antigen-sensitive cells (8). Marrow "lymphocytes' fail to con-
fer immunological competence to thymectomized-irradiated mice (9) and
may, furthermore, represent a heterogeneous cell population (10). We
.
consider the observed correlations consistent with the possibility that cer-
tain, if not all marrow "lymphocytes" are erythroid precursor celle, al-
though we are aware that direct conclusive evidence for the hypothesis is
still lacking.
Materials and Methods
Mice, irradiation and other treatments. (C3H/ Anf ® C57B1)F,/Cum female
mice, 12-15 weeks of age wore used throughout. X radiation was adminis-
REAREHE
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tered to prospective marrow recipients by a 300 KV (peak) Maxitron X ray
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unit, HVL of 0.5 mm of Cu, at an exposure rate of ~ 70 R per minute. Mice
received 800 to 900 R of whole body X radiation in revolving partitioned lu-
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.
cite cages.
Polycythemia was induced in recipients of marrow by hypertransfu-
sion of isogenic washed red blood cells to suppress erythropoiesis. Prospec-
tive blood donors were exposed to 1000 R of whole body X radiation to inac-
tivate circulating stem cells.
Prospective donors of marrow were given in some experiments
cortisol acetate to reduce the number of marrow "lymphocytes." The drug
was suspended in saline and Tween 20 and administered intraperitoneally
in four successive days. Two mg of cortisol were given with each of the
first two injections, and 1 mg with each of the last two. Marrow was har-
vested 9 days after the initial injection.
Cell suspensions. Marrow cells were obtained from femurs and tibias by
R
VY
by:
ALL Ant
flushing the bones with chilled Tyrode's solution. Cell suspensions were
filtered through a 200 mesh/inch stainless steel cloth and nucleated cells
were counted in a Coulter electronic particle counter. For the purpose of
varying the frequency of cell types in marrow, aliquots of cell suspensions
were filtered through columns of packed glass wool (11). The columns ad-
sorb preferentially, but not exclusively, the larger cells of the marrow.
Consequently, the relative number of small and ri.edium-sized "lymphocytes"
1
.
increases in the filtered cell suspensions. Aliquots of the final suspensions
.
2
24:
.
7,.
.
were centrifuged for 10 minutes at 1800 g in a table centrifuge and the re-
sulting pellet was smeared on glass slides with a sable brush for differen-
tial cell counts. The cells were stained with benzidine for hemoglobin and
counter-stained with Wright-Giemsa stain to facilitate distinction between
small erythroid cells and "lymphocytes."
Assays for hematopoietic cell production by transplanted marrow. Appro-
priate numbers of marrow cells from a single preparation were transplanted
into one or two groups of recipients: (a) irradiated mice made polycythemic
by hypertransfusion of red blood cells, and (b) irradiated mice given Tyrode's
solution instead of erythrocytes. At varying intervals thereafter, 0.5 uc of
131
5-iodo-2'-deoxyuridine-1311 (hereafter abbreviated "IUDR) was injected
into the peritoneal cavity of each animal to label the cells engaged in syn-
thesis of DNA. IUDR is a thymidine analog incorporated specifically into
the DNA of mouse cells (12). One hour prior to the injection of "SIUNR each
animal was also given intraperitoneally 10-7 moles of 5-fluro-2'-deoxyuridine
for the purpose of inhibiting the endogenous formation of thymine precursors
which compete with "SIUNR for incorporation into DNA. Unincorporated
131
I radioactivity is excreted from the regenerating hematopoietic tissues
131
within 12 hours. Therefore, mice were killed 18 hours after injection of
the label. in some instances, mice were given a mixture of 1 mc of "IUDR
and 0.1 MC of 59Feci, to label cells engaged in synthesis of DNA and hemo-
globin. The two radioactive labels do not interact nor interfere with the
processes governing their incorporation into regenerating hematopoietic
1. NYT
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TTIK. TOUT ETT
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8.
tissue (unpublished observations). Radioactivity retained in the spleens was
measured individually in a well-type crystal scintillation counter and ex-
pressed as percentage of the radioactivity injected into the animals, above
the percentage of splenic retention of radioactivity in appropriate control
'mice not injected with marrow. The splenic uptake of 13 IUDR estimates,
therefore, the extent to which stem cells of the grafted marrow are capable
of producing DNA synthesizing, i. e., dividing hematopoietic cells. In poly-
cythemic recipients such cells are known to be non-erythroid because of
their inability to incorporate "Fe and because of their rnorphological char-
acteristics (13). The splenic uptake of 59 Fe estimates, on the other hand,
the extent to which stem cells of the same marrow inoculum are capable of
producing hemoglobin synthesizing, i.e., erythroid, hematopoietic cells.
In experiments designed to measure the number of colony-forming
cells in donor marrow, recipients were killed 9 days after transplantation.
Their spleens were fixed in Bouin's solution and the total nurnber of colonies
grossly visible on the surface of each spleon was counted.
Results
Growth of transplanted marrow as affected by hypertranfusion polycythemia.
One million nucleated marrow cells were infused into three groups of irradi-
ated recipients: (1) non-polycythemic mice; (2) polycythemic mice trans-
fused with erythrocytes on the day of marrow transplantation and on days 2,
4, and 6 thereafter; (3) non-polycythemic mice at the time of transplanta-
. 9.
tion but transfused with erythrocytes on days 3, 4, and 6. Mice exposed to
X rays - but not grafted with marrow - served as radiation controls and re-
ceived either erythrocytes or Tyrode's solution. Growth of the transplanted
marrow in the spleens of recipients was estimated at daily intervals (Fig. 1).
The expansion of celle synthesizing DNA and/or hemoglobin was exponential
between day 3 and 5 in non-polycythemic mice, with doubling times ranging
from 10 to 12 hours. The expansion of cells synthesizing DNA, but not hemo-
globin, was also exponential in recipients made polycythemic at the begin-
ning of the experiment, but the doubling time was 35. 7 hours long. It has
been verified that the polycythemic state did not interfere with "SIUDR
utilization by determining labeling indexes on radioautographs of spleen and
marrow smears from animals injected with SH-IUDR instead of "SIUAR.
It has also been verified that the uptake of radioactive precursors in the
spleens of both types of recipients was a linear function of the dose of
grafted cells at days 5, 6, and 7 after transplantation (unpublished results).
The relative size of the proliferating cell pool appears, therefore, to be
considerably smaller in the spleens of the polycythemic mice because of the
absence of erythroid cells, established by functional (Fig. 1B) as well as
histological criteria, and because of the longer doubling time of non-eryth-
roid cells. When polycythemia was induced in marrow recipients three days
after transplantation, the expansion of cella synthesizing DNA and/or hemo-
globin slowed 24 to 48 hours later, presumably, because erythropoiesis was
being suppressed (Fig. 1). Subsequently, the expansion of dividing cells not
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- 10 -
want them the
synthesizing hemoglobin became indistinguishable from the expansion of
the same cell types in recipients maintained polycythemic from the begin-
ning of the experiment, Hence, the proliferation of non-erythroid cells in
the spleens of the latter group of marrow recipients was not detectably
affected, neither by the full erythropoietic activity which went on during
the three days prior to erythrocyte transfusion, nor by the suppression of
erythroid cell production from day 4 to 7. :
The effect of glass wool filtration on marrow stem cell functions. We have
reported that a given number of nucleated cells from filtered marrow was
more efficient than the same number of cells from the original whole mar-
row in repopulating recipient spleens with dividing hematopoietic cells (11).
In the following experiment three stem cell functions of filtered and unfiltered
ay.
ALLE
marrow were assayed by transplantation, namely, the number of colony-
forming cells, the production of cells synthesizing DNA, and the production
w
4
of cells synthesizing hemoglobin. The two latter cell populations overlap
considerably, although a minority of cells divide without synthesizing hemo-
globin and vice versa (Fig. 1). Appropriate numbers of marrow cells were
.
infused into irradiated recipients and killed 6 or 9 days later, depending on
the method of assay (Table 1). Filtered marrow was more efficient than un-
filtered marrow in promoting splenic uptake of 1311UAR and of 59 Fe. Fur-
thermore, it contained more colony-forming cells (Table 1). Concentration
TAMBWE
of precursor cells by glass wool filtration can be expressed by the ratio
between the measured functions (per unit number of grafted cells) of the
. 11.
filtered and the corresponding unfiltered marrow suspensions. The ratio
will be designated hereafter as F/U coefficient. In this experiment the F/U
coefficients for the three stem cell functions assayed were similar. Figure
2 is a frequency distribution diagram of F/U coefficients for the ability of
marrow to repopulate recipient spleens with hematopoietic dividing cells.
The total number of experiments reported is 23 with a mode F/U coeffi-
cient value between 1.5 and 1.8.
Filtered and unfiltered marrow was also transplanted into groups
of irradiated polycythemic and non-polycythemic hosts to establish whether
the frequency of precursors of non-erythroid cells, i. e., of cells engaged in
synthesis of DNA but not of hemoglobin, would also increase upon filtration.
The values of F/U coefficients obtained in four experiments of this type are
reported in Table 2. The ability of marrow to promote splenic uptake of
"
IUR and of "Fe in non-polycythemic hosts was increased in all instances
as is indicated by F/U coefficients greater than 1. The absolute values of
the coefficients were similar within each experiment. This was expected on
the basis of previous experience (Table 1 and Fig. 2) and because the majority
of cells in regenerating spleens incorporate both radioactive precursors. In
contrast with these results, the filtered marrow Buspensions were not superior
to unfiltered marrow in repopulating spleens of polycythemic hosts, as is
indicated by F/U coefficients of 1 or less than 1. In one of the four experi-
ments (No. 4 of Table 2) the F/U coefficient in polycythemic hosts was 1.29.
23
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Nevertheless, the experiment is consistent with the other three because the
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- 12 -
F/U coefficient for production of non-erythroid cells was significantly lower
than the coefficients for erythroid and total hematopoietic cell production in
non-polycythemic hosts (Table 2). Thus, the results of glass wool filtration
experiments indicated, as did the marrow growth curves of Figure 1, that
production of non-erythroid cells by grafted marrow is not affected by changes
in the marrow content of erythroid precursors assayed by the "STIUDR and
Fe techniques. More interestingly, non-erythroid cell production seemed
also unaffected by changes in the marrow's content of colony-forming cells
.
.
.
.
which are thought to be pluripotential (4-6).
.
.
.
.
.
.
.
.
We have reported that upon filtration of marrow the frequency of
. i
r
2
small and medium-sized "lymphocytes'' and of stem cells increased to the
same extent (11). Results of the 23 filtration experiments described herein
fully confirm our earlier report. Figure 3 is a linear plot of the calculated
F/U coefficients for marrow "lymphocytes' against the F/U coefficients for
stem cell functions. If marrow "lymphocytes" were stem cells, the F/U
coefficient values for the pertinent stem cell function or functions should
-
.
.
cluster about a regression line with a slope of 1. Otherwise, the coeffi-
-
cient values should either be irregularly distributed or cluster about a hori-
zontal line. The plot is consistent with the hypothesis that marrow "lympho-
cytes are precursors of erythroid cells but not of non-erythroid hemato-
poietic cells. None of the other cell types which we have classified in mar-
row yielded similar results. We list in Table 3 the T/U coefficient values
calculated for all cell types in marrow suspensions employed in two separate
e non-
- 13.
experiments; the corresponding stem cell functions and their F/U coeffi-
cients are described in Table 2, experiments 1 and 4.
It has been observed that administration to mice of cortisol reduced
the frequency of "'lymphocytes" without reducing the number of stem cells
in marrow (10). The observation suggested that not all marrow "lympho-
cytes'' were alike either with respect to sensitivity to lympholytic action of
interest, therefore, to filter through glass wool columns marrow suspen-
sions harvested from donors treated with cortisol and to establish (a) whe-
ther such filtered marrow was more efficient than unfiltered marrow in
repopulating recipient spleens, and (b) whether induced changes in fre-
quency of "lymphocytes'' apparently resistant to cortisol treatment, still
would correlate with the changes in stem cell functions. Three experiments
of this type are reported in Table 4; in each case cortisol reduced the per-
centage of "lymphocytes' in donor marrow to 8-11% (normal values range
from 20 to 25%). Upon filtration, the frequency of stem-cells and of
"lymphocytes' resistant to cortisol increased, yielding comparable F/U co-
efficients. The results are, therefore, consistent with those obtained by
.
as saying marrow from untreated donors, illustrated in Figure 3.
Discussion
The relevant findings of the present investigation can be summarized
as follows:
• 14.
bwp-
(a) Apparently, no pluripotent hematopoietic stem cells are shunted
from or into the production of differentiating non-erythroid cells as a con-
a
sequence of experimentally induced cessation of erythropoietic activity.
(b) Precursors of erythroid cells and colony-forming cells are con-
:*
.
...
centrated in the effluent from glass wool columns upon filtration of marrow
H
-
whethe
suspensions. Precursors of non-erythroid cells are not concentrated by the
same procedure.
(c) The competence of transplanted marrow to initiate erythropoiesis -
but not leukopoiesis - appears to be a function of the number of "lymphocytes"
present and not of other cell types. Marrow "lymphocytes" may be hetero-
geneous because cortisol reduces their number without reducing that of stem
cells. Hence, a subpopulation of "lymphocytes" may provide the stem cells
leading to erythroid cell production.
Differentiation of erythroid independently of non-erythroid cells in
marrow chimeras, as described here, is in line with the findings of several
other investigators. It has been demonstrated by a variety of methods that
self-renewal of stem cells and of erythropoietin-sensitive cells (14-17),
granulocytopoiesis, platelet production, and erythropoiesis (18-21) may
proceed independently. All these findings and those summarized under (a)
are compatible with, but not proof for, the interpretation that independent
control mechanisms of hematopoietic proliferation and differentiation act on
a primitive pluripotent stem cell of marrow, which is assayed by transplanta-
tion (4, 15). In contrast, our findings summarized under (b) describe an
- 15 -
experimental condition in which colony-forming cells and precursors of
erythroid cells are concent:ated in vitro, while precursors of non-eryth-
roid cells remain quantitatively unchanged. We, therefore, conclude that
we are assaying primitive, independent "precursor" cells with limited
potential by the short-term follow up of transplanted marrow from adult
mice. Such cells are capable of differentiation only into one of the major
hematopoietic lines, but may, nevertheless, be self-replicating, i. e., true
stem cells. Preliminary unpublished results of current investigations sug-
just that the precursors of erythroid cells assayed by the 131 IUDR - 59Fe
technique and concentrated by glass wool filtration are indeed unipotential
stem cells.
Unipotential stem cells descend from more primitive, rare, pluri-
potent elements which, presumably, are also present in the marrow of adult
mice. The existence of such a stem cell is generally accepted on the basis
of theorctical expectations and experimental findings. The evidence for the
clonal nature of colonies (6) and for the pluripotential function of colony-
forming cells (4-6) conflicts with our present observation that colony-forming
cells concentrated by glass wool filtration are not the precursors of granulo-
cytic cells. Perhaps, the property of colony formation is shared by primi-
tive hematopoietic cells of more than one kind, namely, by rare pluripoten-
tial and by more abundant unipotential (erythropoietic?) stem cells. Similar
views have been expressed by Curry, Trentin and Wolf (19). Enumeration
of colony-forming cells of filtered and of unfiltered marrow assayed by
. 16.
transplantation into polycythemic and non-polycythemic recipients should
r
yield the decisive information needed to answer: this question.
e
There is no doubt that erythropoietic competence and frequency of
.vi
:1
With
marrow "lymphocytes" are related. Other investigators have described
h
similar correlations between hematopoietic competence of mouse marrow
-'
**
W
. -
and its content of "lymphocytes" (22, 23). The findings strongly suggest
.
.
.
that marrow cells with the morphologic features of the lymphocyte are pri-
..
.
.
.
mitive hematopoietic cells in the mouse.
.
*
.
.
The types of experiments presented here focus attention on mouse mar-
!
H
-
.
row "lymphocytes'' as erythroid stem cells and indicate a direct experimental
.**
.
approach to a problem which has been of concern to many hematologists in
the past (24, a review) and to several participants in this symposium.
Summary
The growth of hematopoietic cells derived from grafted marrow was
estimated in the spleens of irradiated recipient mice by labeling dividing cells
with the radioactive DNA precursor 5-iodo-2'-deoxyuridine-5-1. Erythro-
poiesis is suppressed in marrow recipients made polycythemic by hyper-
transfusion with erythrocytes; in this case the dividing cells are non-eryth-
roid in nature, i.e., they do not synthesize hemoglobin. Erythropoiesis was
estimated separately by labeling the recipient spleens also with 59 Feciz.
Polycythemia induced either at the time of marrow transplantation or
three days later reduced the pool of dividing hematopoietic cells by an amount
Bleri
clearly related to the suppression of erythropoiesis. There was no evidence
. 17.
for the shunting of pluripotent stem cells from or into the production of non-
crythroid cells as a consequence of the induced changes in erythropoiesis.
Filtration of inarrow through columns of glass wool increased the concen-
tration of colony-forming cells and of cells capable of establishing erythro-
poiesis in irradiated recipients; the procedure, however, did not concen-
trate precursors of leukocytic elements. The results indicate that primi-
tive, independent hematopoietic cells with limited potential, as opposed to
pluripotent stem cells, were assayed by transplantation of marrow from
adult donor mice. The data also suggest that colony-forming cells are not
all pluripotent but, rather, that under these conditions most of them are unre-
lated to the production of non-erythroid hematopoietic cells. The frequency
of marrow "lymphocytes" was increased in the effluent from glass wool
columns and was consistently correlated with erythroid precursor function.
This correlation was observed without exception in the twenty-three separate
experiments described. The concentration of other cell types in marrow
decreased upon filtration. Hence, marrow cells, resembling the peripheral
lymphocyte morphologically but not functionally may be the primitive eryth-
ropoietic cells assayed by our transplantation and filtration technique.
Acknowledgements
Statistical analysis of the data has been performed by Dr. D. G.
Gosslee, Mathematics Panel, Oak Ridge National Laboratory. The participa-
tion of one of us (G. C. ) in this Symposium has been supported by USPHS
Contract PH-43-65-44 (Roswell Park Memorial Institute).
.18 -
Literature Cited
1. Cudkowicz, G., Upton, A. C., Smith, L. H., Gosslee, D. G., and
.-.
-
Hughes, W. L. - An approach to the characterization of stem cells in
-
.
.
.
mouse bone marrow. Ann. N. Y. Acad. Sci., 114: 571-582, 1964.
2. Smith, L. H. - Marrow transplantation measured by uptake of 59 Fe
by spleen. Am. J. Physiol., 206: 1244-1250, 1964.
3. Gurney, C. w., Lajtha, L. G., and Oliver, R. - A method for inves-
tigation of stem-cell kinetics. Brit. J. Haemat., 8: 461-466, 1962.
4. Siminovitch, L., McCulloch, E. A.and Till, J. E. - The distribution
of colony-forming celis arnong spleen colonies. J. Cell. Comp. Physiol.,
5. Till, J. E., and McCulloch, E. A. - A direct measurement of the
radiation sensitivity of normal mouse bone marrow cells. Rad. Res.,
14: 213-222, 1961.
6. Becker, A. J., McCulloch, E. A., and Till, J. E. - Cytological demon-
stration of the clonal nature of spleen colonies derived from transplanted
mouse marrow cells. Nature, 197: 452-454, 1963.
7. Friedman, H. - Distribution of antibody plaque forming cells in various
tissues of several strains of mice injected with sheep erythrocytes.
Proc. Soc. Exptl. Biol. Med., 117: 526-530, 1964.
8. Kennedy, J. C., Siminovitch, L., Till, J. E., and McCulloch, E. A. -
A transplantation assay for mouse cells responsive to antigenic stimula-
tion by sheep erythrocytes. Proc. Soc. Expti. Biol. Med., 120: 868-
873, 1965.
. 19.
9. Cross, A. M., Leuchars, E., and Miller, J. F. A. P. - Studies on
the immune response in irradiated mice thymectomized in adult life.
J. Exptl. Med., 119: 837-850, 1964.
10. Bennett, M., and Shearer, G. M. - Stem cells and "lymphochtes" in
mouse bone marrow. Fed. Proc., 24: 240, 1965 (abstract).
11. Cudkowicz, G., Bennett, M., and Shearer, G. M. - Pluripotent stem
cell function of the mouse marrow "lymphocyte." Science, 144: 866-
868, 1964.
12. Hughes, W. L., Commerford, S. L., Gitlin, D., Krueger, R. C.,
Schultze, B., Shah, V., and Reilly, P. - Deoxyribonucleic acid metabo-
lism in vivo: I. Cell proliferation and death as measured by incorpor-
ation and elimination of iododeoxyuridine. Fed. Proc., 23: 640-648,
1964.
13. Jacobson, L. O., Goldwasser, E., and Gurney, c. W. - Transfusion-
induced polycythemia as a model for studying factors influencing erythro-
poiesis. In Ciba Foundation Symp. on Haemopoiesis. J. & A. Chur-
chill, London, 1960, p. 423-445.
14.
Bruce, W. R., and McCulloch, E. A. - The effect of erythropoietic
stimulation on the hemopoietic colony-forming cells of mice. Blood,
23: 216-232, 1964.
15. Till, J. E., Siminovitch, L., and McCulloch, E. A. - Growth and dif-
ferentiation of marrow cells transpianted in anernic and plethoric mice
of genotype w/w'. Exptl. Hematol., 2: 59, 1966 (abstract).
K
AZ
ft
PT
..
...
FMRY
RES
. 20.
16. Fried, w., Weisman, M., and Gurney, c. W. - Comparison of the
rate of recovery of CFU and ERC in mice after 200 R total-body X-
irradiation. Exptl. Hematol., 9: 10-13, 1966.
17. Schooley, J. C., Cantor, L. N., and Havens, V. W. - Relationship
between growth of colony-forming cells and erythropoietin-sensitive
cells in mice irradiated with 200 R of 6°co gamma rays. Exptl.
Hematol., 2: 55-56, 1966 (abstract).
18. Liron, M. , and Feldman, M. - The specific suppression of the differ-
entiation of erythroid clones in polycythemic animals. Transpl., 3:
509-516, 1965.
19. Curry, J., Trentin, J., and Wolf, N. - Control of spleen colony his-
tology by erythropoietin, cobalt, and hypertransfusion. Exptl. Hematol.,
7: 80, 1964 (abstract).
NATALANA
20. Alexanian, R.' - Specificity of erythropoietin-sensitive "stem cells" for
red blood cell production. Exptl. Hematol. , 7: 86, 1964 (abstract).
-
-
21. Blackett, N. M., Roylance, P. J., and Adams, K. - Studies of the capa-
city of bone-marrow cells to restore erythropoiesis in heavily irradiated
VRAWO
rats. Brit. J. Haemat., 10: 453-467, 1964.
22. Brecher, G., Endicott, K. M., Gump, H., and Brawner, H. P. -
Effects of X-ray on lymphoid and hemopoietic tissues of albino mice..
Blood, 3: 1259-1274, 1948.
.
ENT,
13
23. Kurnick, N. B., and Nokay, N. - Repopulation of bone marrow in mice:
-6,
number and type of cells required for post-X-irradiation protection.
nr..
- 21 -
Rad. Res., 25: 53-67, 1965.
24. Yoffey, J. M. - The lymphocyte. Annual Review of Medicine, 15: 125-
148, 1964.
1
.
I
.
ithin time
wir whole
w
Table 1
The Effect of Glass Wool Filtration on
Stem Cell Functions of Grafted Marrow
Unfiltered
Filtered
· F/U
Method of Assay
Marrow
Marrow
Coefficient
1.69
Uptake of ***IUDR*
Uptake of 59 Fe*
Nodular Colonies+
0.69 3.05 (5)*
4.22 + .41 (5)
4.68 3.23 (23)
1.17 6.13 (6)
8.29 * 1.43 (6)
7.79 #.51 (17)
5
(5)
1.96
1.67
* Mcan splenic uptake (% of injected amount) and S. E. per 100 grafted cells 6
days after transplantation.
Mean number of colonies per spleen and S. E. per 10* grafted cells 9 days
after transplantation.
Number of recipient mice in parenthesis.
Table 2
Differential Effect of Glass Wool Filtration on Erythropoietic
And Non-erythropoietic Stem Cell Functions
F/U Coefficients
Non-polycythemic Hosts
Polycythemic Hosts
13 IUDR Uptake
59Fe Uptake
i 31
IUDR Uptake
Exp. 1
:
1.41
1.30
0.91
Exp. 2
1.51
0.94
Exp. 3
1.68
1.70
:
:.
0.90
1.29
Exp. 4
1.69
1.96
L
.
2
-'.
-,
Table 3
The Effect of Glass Wool Filtration on the Frequency of Cell Types
In Marrow, as Indicated by the F/U Coefficient Values
F/U Coefficients
Cell Type
Exp. 1*
Exp. 4*
Lymphocytes:
Small and medium-sizeá
1. 42
1.78
Large
0.19
0.50
Granulocytes
1.27
0,65
Metamyelocytes
0.83
0.84
-
...
Myelocytes
0.25
1.11
-
.
-
Promyelocytes
0.61
i
w
Myeloblasts
0.84
0.73
bi
Normoblasts
0.76
3. 24
it. Im
Erythroblasts
0.13
0.55
Proerythroblasts
0.06
0.15
Blasts
0.16
0.33
Monocytes
0.34
1.00
Reticulum cells
0.17
0.46
Histiocytes
0.00
0.25
SVL
Plasma cells
0.12
0.38
* Stem cell functions of these marrow suspensions are ce-
ported in Table 2.
!
1.
:
Table 4
Increased Frequency of Stem Cells in Marrow from Donors Treated
With Cortisol Obtained by Filtration Through Glass Wool
..-.
F/U Coefficients For
-.
Frequency of "Lymphocytes"
In Unfiltered Marrow (%)
"Lymphocytes'*
Stem Cells**
8.27
2.43
2. 33
8.69
1.72
1.60
11.98
1.59
1.64
* Method of assay: morphological
** Method of assay: splenic uptake of "
IUDR in non-polycythemic hosts.
Figure Legends
Figure 1. The expansion of hematopoietic cells in the spleen of irradiated
recipient mice following transplantation of 106 nucleated marrow cells.
Growth was estimated in Figure 1A by the uptake of "STIUAR (dividing cells ·
of mixed types) and in Figure 1B by the uptake of "Fe (hemoglobin synthe-
sizing erythroid cells). Doubling times (tz) are indicated in hours with 95%
confidence limits in parentheses.
Onon-polycythemic throughtout
O
polycythemia initiated on day O and maintained throughout
A-
A polycythemia initiated on day 3 and maintained thereafter.
Figure 2. Frequency diagram of the efficiency of glass wool filtration of
7
marrow (F/U coefficients). Stem cell content of suspensions was assayed
by the uptake of 13AIUR in recipient spleens. Total number of observations
23.
Figure 3. Correlation between frequency of marrow "lymphocytes" and cer-
tain stem cell functions. F/U coefficients should cluster about a line with
slope of 1 (dotted regression line) if marrow "lymphocytes" were stem cells,
F/U coefficients should cluster about a line with slope of 0 (dotted horizontal
line) or scatter at random if "lymphocytes were unrelated to stem cell func-
tions.
"SIUDR uptake in non-polycythemic hosts - 23 experiments
A
Fe uptake in non-polycythemic hosts - 3 experiments
Nocular colonies in non-polycythemic hosts - l experiment
L.
O
131
IUDR uptake in polycythemic hosts - 4 experiments
*
A
.
.
NET

f


2.00,
-
to 12.3 HRS
cias-144)
te 9.77 HRS
10.76 - 11.01
tieteni
..
FE UPTAKE (%)
w 2.00
MEAN SPLENIC 131IUDR UPTAKE (%)
136.7 HRS
(32..-30.11
-
3,20
.
*
.
P
.
M
.
. .
MEAN SPLENIC
0.50
8.00-od
* 0.207
1
0.01
0.10.
.
"
6
6
7
3
4
5
6
7
.
..
ir...'.
I
DAYS AFTER MARROW TRANSPLANTATION
.-

-
"
-
r
-
.
1
-
- -
.
.
F
.
,

NUMBER OF
OBSERVATIONS
1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6
FILTERED
UNFILTERED COEFFICIENT
-
.
Fij, 2
- --- ---.--..
731 79 A
r
.
.
FIU COEFFICIENT FOR STEM CELL FUNCTIONS

.
.
...
..
......
.
1.4 1.8 2.2 2.6 3.0
COEFFICIENT FOR "LYMPHOCYTES"
...
Fin3
.
1864184. '. .
som
en annan song
wow.component
..
END



DATE FILMED
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SW



"
"