Stem Cells, Vol. 17, No. 4, 219-225,
July 1999
© 1999 AlphaMed Press
A Simple, One-Step Clonal Assay Allows the Sequential Detection of Committed (CFU-GM-like) Progenitors and Several Subsets of Primitive (HPP-CFC) Murine Progenitors
Zoran Ivanovi
a,
Benedetta Bartolozzib,
Pietro Antonio Bernabeib,
Maria Grazia Cipolleschia,
Pavle Milenkovicc,
Vincent Pralorand,
Persio Dello Sbarbaa
a Department of Experimental Pathology and Oncology, University of Florence, Florence, Italy;
b Department of Hematology, A.O. Careggi, Florence, Italy;
c Institute for Medical Research, Belgrade, Yugoslavia;
d Laboratory of Experimental Hematology, University of Limoges, Limoges, France
Key Words. HPP-CFC • IL-3 • G-CSF • In
vitro • Mouse • Bone marrow
Dr. Persio Dello Sbarba, Dipartimento di Patologia e Oncologia Sperimentali, Università di Firenze, viale G.B. Morgagni 50, I-50134 Florence, Italy.
 |
ABSTRACT
|
|---|
Murine bone marrow (BM) cells were cultured in semisolid medium
containing interleukin 3 (IL-3) and high doses of G-CSF. Colonies were
counted twice, at day 7 and day 14, and the number of
granulocyte/macrophage colony-forming units (CFU-GM) accurately
estimated by the subtraction of day-14 from day-7 colonies, based on
the principle that colonies detectable at day 7 and persisting beyond
day 14 are generated by significantly more immature progenitors. The
frequency of colonies relative to their size was determined and used
to define subsets of high proliferative potential colony-forming cells
(HPP-CFC). Two main groups of HPP-CFC were considered: those
generating colonies of 0.6-1.8 mm of diameter or larger than 1.8
mm. The characterization of these groups showed that they correspond
to different functional subsets of HPP-CFC. The replating ability of
colonies was estimated. The percentage of clonogenic progenitors in
the S phase of cell cycle was measured by cytosine arabinoside suicide
assay. The sensitivity of colonies to 5-fluorouracil (5-FU) in vitro
was determined and their survival after an in vivo treatment with 5-FU
compared with that of colony-forming units in spleen (CFU-S). This
technique allowed identification of: A) CFU-GM; B) relatively mature
HPP-CFC, probably corresponding to CFU-S day12; C) more primitive
HPP-CFC, relatively resistant to 5-FU in vivo and closely
corresponding to CFU-S day 14, and D) very primitive HPP-CFC,
resistant to 5-FU in vitro. This simple, rapid, and versatile method
allows the detection of a broad range of hematopoietic progenitors in
murine BM, from committed progenitors to largely quiescent, primitive
stem cells, as well as the evaluation of the progenitors'
self-renewal and proliferative potential. Stem Cells
1999;17:219-225
|
Introduction
|
|---|
The identification and quantitation of hematopoietic stem and
progenitor cells in humans and mice is still a question of major
interest for researchers and clinicians. In vivo assays such as spleen
colony formation (CFU-S) [1, 2] or bone marrow (BM) repopulation (MRA) are
available [3, 4]. As these methods require large
numbers of animals, in vitro approaches have been developed. Long-term
cultures (LTC) of hematopoietic cells on BM stromal layers [5] allow the proliferation and
differentiation of very primitive stem cells [2, 6, 7]. However, quantitative assays based on LTC,
such as the measure of LTC-initiating cells [8] or cobblestone area-forming cells
[9] are time-consuming
and their results difficult to compare among different laboratories
due to modifications of the originally described methods [10].
A relatively simple one-step assay allows the detection of high
proliferative potential colony-forming cells (HPP-CFC). This assay,
developed initially using murine [11] and later human [12] BM, is much faster to perform and its
results easier to analyze. HPP-CFC: A) are relatively resistant to an
in vivo treatment of mice with 5-fluorouracil (5-FU) [3]; B) generate macroscopic colonies
in response to various combinations of growth factors, and C) have a
high replating efficiency in secondary assays. Based on their
responsiveness to various combinations of two to several growth
factors, HPP-CFC have been classified as primitive (HPP-CFC-1),
intermediate (HPP-CFC-2), and mature (HPP-CFC-3) [13, 14]. These progenitors are related to
progenitors responsible for MRA, CFU-S/day13 (CFU-Sd13) and CFU-Sd8,
respectively. Methylcellulose cultures have been introduced, allowing
the transfer of colony cells to secondary clonal assays, and thus, the
estimate of the secondary colony-forming efficiency of human
HPP-CFC-derived colonies [15].
Human blood HPP-CFC grow in the presence of very high doses of
G-CSF [16] and murine
HPP-CFC in the presence of interleukin 3 (IL-3) plus relatively low
doses of G-CSF [17]. The
culture system described here is based on the combination of both
high-dose G-CSF and IL-3 with methylcellulose cultures of murine BM
cells (BMC). In these cultures, we characterized the size of colonies,
their kinetics of appearance and development, and their secondary
colony-forming efficiency. The percentage of primary clonogenic
progenitors in the S phase of cell cycle and the sensitivity of
HPP-CFC to an in vitro incubation with 5-FU were also
estimated. Finally, the percentage survival upon in vivo treatment
with 5-FU of HPP-CFC was compared with that of CFU-S. The measure of
these parameters, taken together, resulted in the development of a
simple culture system allowing the sequential detection of committed
and primitive clonogenic progenitors.
|
Materials and
Methods
|
|---|
Preparation of BMC Suspension
BMC were recovered from 10- to 14-week-old CBA mice by injection
of RPMI-1640 medium (GIBCO Ltd.; Paisley, UK) into femoral bone
shafts, washed, and resuspended in the same medium. Viable cells were
counted in a hemocytometer, after suspension in phosphate-buffered
saline (PBS) containing 4% acetic acid and 0.03% Trypan
blue.
In Vitro Colony-Formation Assay
BMC were plated in a premixed methylcellulose culture medium
(Methocult H4230, StemCell Technologies Inc.; Vancouver, Canada),
adjusting the final concentrations of methylcellulose to 0.9% and of
fetal bovine serum to 30%. Recombinant murine IL-3 (PeproTech EC;
London, UK) and recombinant human G-CSF (Neupogen; Dompé
BioTec; Milan, Italy) were added at 10 ng/ml and 500 ng/ml,
respectively. In preliminary experiments, these concentrations
provided the highest number and replating ability of primary
colonies. BMC suspensions (0.25 ml) were added to the complete culture
medium (2.25 ml), vortexed, and distributed in Petri dishes (Falcon,
Becton Dickinson; Meylan, France; 1 ml/dish). Cultures were incubated
in a water-saturated, 5% CO2
atmosphere.
Counting of In Vitro
Colonies
Colonies (>50 cells) were counted at day 7 and cultures
returned to the incubator until day 14, when colonies were counted
again. The number of colonies derived from committed progenitors
(CFU-GM) was obtained by subtracting the number of colonies scored at
day 14 from the number scored at day 7. The colonies still present at
day 14 (developed from primitive progenitors) were scored and
classified according to their diameter: group 1, diameter 0.6-1.8 mm;
group 2, diameter >1.8 mm.
Replating of Cells
From Primary In Vitro Colonies
Day-14 colonies harvested from methylcellulose cultures were
resuspended in 200 µl of RPMI-1640 and cells counted and replated
in secondary methylcellulose cultures established as above. Secondary
colonies were scored at days 7 and 14. The same procedure was used to
establish tertiary cultures.
Spleen Colony-Formation
Assay
This assay was performed according to the method of Till
and McCulloch [1], with the modifications described in detail
in a previous study [18]. Donor BMC were transplanted into lethally
irradiated (x-rays, 9 Gy, 0.96 Gy/min; Philips RT-305 source)
syngeneic mice (8-12 mice per group; 4 ¥ 104 cells per
mouse). Recipients were killed 8, 12, or 14 days after
transplantation, their spleens removed and fixed in
Telleyesniczky's solution, and the colonies counted
(counts of CFU-Sd8, CFU-Sd12, and CFU-Sd14,
respectively).
Cytosine Arabinoside (Ara-C) Suicide
Assay
BMC were incubated (106 cells/ml) for 1 h in RPMI-1640
supplemented with 20% horse serum (HyClone; Logan, Utah) in the
presence or absence of 20 µg/ml of Ara-C (Roche; Neuilly-sur-Seine,
France), an S-phase-specific cytotoxic drug. Cells were then washed
three times in PBS and plated at 2 ¥ 104 cells/ml in
methylcellulose-containing medium prepared as above. Colonies were
scored at days 7 and 14. The ratio of the colony-formation efficiency
of Ara-C-treated cells to that of untreated cells represents the
percentage of progenitors in the S phase of cell cycle [18].
Determination of HPP-CFC
Sensitivity to 5-FU In Vitro
BMC were incubated (106 cells/ml) for 24 h in RPMI-1640
supplemented with 20% HS, in 25 cm2 culture flasks
(NUNC-International; Hereford, UK; 10 ml/flask), in the presence or
the absence of 10 µg/ml 5-FU (Roche), a cell-cycle-specific
cytotoxic drug. Cells were then washed three times in PBS, counted,
and plated in methylcellulose cultures established as above (5 ¥
104 cells/dish). Colonies were scored at days 7 and
14.
Determination of the In Vivo Effects of 5-FU on
HPP-CFC and CFU-S
Mice were injected i.v. with 5-FU (150 µg/g), dissolved in 0.2
ml PBS [19] or 0.2 ml
PBS (controls) and sacrificed 48 h later. BMC recovered from each
animal (six mice per experimental condition) were counted individually
as described above and then pooled to concentrations suitable for
plating in methylcellulose cultures or transplantation into lethally
irradiated mice. Colonies developed in culture or in spleen were
counted at days 7 and 14 and at days 8, 12, and 14, respectively. The
percentage of 5-FU-resistant progenitors was obtained from the ratio
of the colony-formation efficiency of BMC recovered from 5-FU-treated
animals to that of control animals.
Statistical
Analysis
The differences between variables were evaluated by Student's
t-test for independent
samples.
|
Results
|
|---|
Characterization and classification of colonies. In
methylcellulose cultures of BMC supplemented with IL-3 and G-CSF, more
than 95% of colonies appeared between day 3 and day 7. From day 7 to
day 14, the number of colonies significantly decreased, while the size
of remaining colonies increased. No colony appeared after day 14. The
colonies that disappeared before day 14 were classified on the basis
of their size, morphology, and cell content as derived from
CFU-GM. Their incidence ( Table
1) was linearly related to the number of plated cells (not
shown).
The diameter of colonies detectable at day 14 varied from 0.2 mm
to 3.6 mm. Since a linear relationship between the number of plated
cells and the number of day-14 colonies was observed only for colonies
larger than 0.6 mm (not shown), small colonies (<0.6 mm) were
ignored. Colonies >0.6 mm were counted and classified into groups
differing by 0.1 mm of diameter (Fig. 1). The stepwise increase of incidence observed
between colonies smaller and larger than 1.8 mm in diameter led us to
distinguish two subsets of day-14 colonies: 0.6-1.8 mm and >1.8
mm. The colonies 0.6-1.8 mm had a lower incidence than day-7 CFU-GM
( Table 1) and contained
mainly macrophages, sometimes also neutrophils (not shown). The
colonies >1.8mm, detected as frequently as those 0.6-1.8 mm
( Table 1), were
macrophage colonies with a densely packed central area and contained
from ~40,000 to ~350,000 cells (not shown). These are typical features
of HPP-CFC-generated colonies [13]. Stained cytocentrifugates showed that most
cells were multinucleated (not shown), as previously described for
HPP-CFC colonies derived from human cord blood cells [15]. These colonies persisted in
culture and continued to grow for three months.
View larger version (17K):
[in this window]
[in a new window]
|
Figure
1. Distribution of day-14 colonies in function of their
diameter. Histograms represent the relative frequency of
colonies in function of their size. Groups differ by 0.1 mm colony
diameter. Data refer to a total number of 225 colonies obtained in
four independent experiments. The dotted line represents cumulative
percentages of colonies. Small colonies < 0.6 mm are not included
in the graph, as they were not linearly related to the number of
plated cells.
|
|
S Phase
Rate of Progenitors
The percentage of each type of progenitor in the S phase of cell
cycle was measured by treating BMC in vitro for 1 h with Ara-C prior
to their plating for clonal assays (Table 1). Approximately 30% of CFU-GM and HPP progenitors
of colonies 0.6-1.8 mm (HPP-CFC 0.6-1.8 mm) were in S phase. By
contrast, only about 3% of HPP-CFC >1.8 mm were in S
phase.
Replating Ability of HPP-CFC >1.8 mm
Colonies
Cells replated from primary colonies >1.8 mm ( Table 2) generated both day-7 and
day-14 secondary colonies. Secondary day-14 colonies still contained
cells able to generate tertiary day-7 colonies. However, the size of
colonies and the clonogenic ability of their cells decreased
progressively from primary to tertiary
cultures.
Progenitor Sensitivity to 5-FU In
Vitro
The effects of a 24-h in vitro pretreatment of BMC with 5-FU on
their colony-forming ability are shown in Table 3. In cultures established with
5-FU-treated cells, the number of both day-7 and day-14 colonies was
very low. As all CFU-GM were cycling, the few day-7 colonies were
still detectable at day 14. The incidence of 5-FU-resistant colonies
(4 ±2 per 105 cells) was 12.5-fold lower than that
of colonies generated from control cells. As all 5-FU-resistant
colonies were about 3 mm in diameter, their replating ability was
compared with that of control colonies having the same diameter and
number of cells. Primary 5-FU-resistant (when compared with control)
3-mm colonies generated comparable numbers of secondary day-7
colonies, 2.3 times more day-14 colonies 0.6-1.8 mm, and a sizable
number of day-14 colonies >1.8 mm, absent in
controls.
Progenitor Sensitivity to 5-FU In
Vivo
In order to better define the hierarchical level of progenitors
detected in our system, we compared their sensitivity to a two-day in
vivo treatment with 5-FU with that of CFU-S day-8, -12, and -14
( Table 4). The percent
survival after 5-FU was 4.0% for CFU-Sd8, 24.2% for CFU-Sd12, and
35.8% for CFU-Sd14. CFU-GM did not survive the treatment, whereas
22.8% of HPP-CFC 0.6-1.8 mm and 36.3% of HPP-CFC >1.8 mm
survived. Thus, the percentage survival was strikingly similar for
CFU-Sd12 and HPP-CFC 0.6-1.8 mm on one side and for CFU-Sd14 and
HPP-CFC >1.8 mm on the
other.
|
Discussion
|
|---|
We describe here a simple experimental system which allows the
sequential detection of committed progenitors as well as of different
subsets of primitive hematopoietic progenitors of the HPP-CFC
family. This system exhibits several original features: A) the use of
high-doses of G-CSF, which has not so far been reported in combination
with IL-3 and for murine BMC, and B) the counting of colonies twice,
at day 7 and day 14, followed by the subtraction of day-14 from day-7
colonies. This procedure provided an accurate estimate of the number
of committed progenitors (CFU-GM), based on the principle that
colonies detectable at day 7 and persisting beyond day 14 are
generated by significantly more immature progenitors. A third
peculiarity of the system is the use of the relative frequency of
colonies versus their size to define subsets of HPP-CFC. Stepwise
increases of incidence between colonies smaller and larger than
certain diameter values were detected. This led to classification of
colonies into three groups: between 0.6 and 1.8 mm, larger than 1.8
mm, and about 3 mm (5-FU-resistant). The characterization of these
progenitor groups showed that they correspond to different functional
subsets of HPP-CFC.
The properties of HPP-CFC subsets were characterized first on the
basis of two main criteria: A) their sensitivity to an in vitro
treatment with 5-FU ( Table
3) and B) the replating ability of the colonies they
generated ( Table
2). 5-FU is a cell cycle-specific agent sparing only
G0 cells, and is therefore suitable for defining a
"resistant" subset of non-cycling cells. 5-FU, indeed,
together with cytokine stimulation, were used to identify human
super-primitive hematopoietic stem cells [19]. Only colonies generated by HPP-CFC >1.8
mm were capable of generating secondary colonies and were in part (8%)
5-FU resistant. All the colonies generated by 5-FU-resistant HPP-CFC
were, in fact, about 3 mm in diameter, so they were compared to 3-mm
colonies generated from control cells (a subset evident in Fig. 1). The 5-FU-resistant
colonies exhibited a far superior capacity to generate secondary
colonies.
Furthermore, to better determine their level in the hematopoietic
hierarchy, HPP-CFC subsets defined according to our system were
compared with progenitors identified by well-established clonal assays
in vivo (CFU-S) and in function of their response to an in vivo
treatment with 5-FU ( Table
4). Significant differences in 5-FU resistance were
observed among CFU-S subsets, confirming the already described
heterogeneity of the CFU-S population [20, 21]. The percentage survival values obtained
for CFU-Sd12 and CFU-Sd14 closely matched those of HPP-CFC 0.6-1.8 mm
and HPP-CFC >1.8 mm, respectively. The latter, in turn,
corresponded very well to the values obtained by Bradley and
coworkers after a 5-FU treatment with the same dose and time-schedule
as in this study [22]. We concluded that HPP-CFC 0.6-1.8 mm and
HPP-CFC >1.8 mm represent quite distinct subsets of
progenitors.
On the basis of all of the above, it was possible to identify four
types of progenitors ( Table
5), characterized as follows: A) committed progenitors of
day-7 colonies (disappeared at day 14), whose incidence, growth
kinetics, percentage in S phase and morphology of cell progeny were
very similar to those of CFU-GM [23]; B) the progenitors of day-14, 0.6-1.8 mm
colonies, apparently corresponding to CFU-Sd12 and more immature than
the HPP-CFC3 of McNiece's classification [14] which include CFU-Sd8; C) the
main subset of the progenitors of day-14, >1.8mm colonies, slowly
cycling (low percentage in S phase), relatively resistant to 5-FU in
vivo, but sensitive to 5-FU in vitro; these progenitors exhibit HPP
and ability to generate secondary and tertiary colonies, but not the
capacity of self-renewal [16], therefore being very similar to CFU-Sd14,
and probably corresponding to the HPP-CFC2 of McNiece's
classification, and D) a minor subset (8%) of the progenitors of
day-14, >1.8 mm colonies, which are mostly non-cycling and thus
resistant to 5-FU in vitro; these colonies generate large numbers of
secondary colonies, 10% of which were >1.8 mm, and their incidence
(4 ± 2 per 105 BMC, Table 3) is similar to that of progenitors
endowed with self-renewal ability within the heterogenous
"CFU-A" population which includes CFU-S and more primitive
stem cells [24,
25], and to that (1-4
per 105 murine BMC) of CAFC day 28 [9], as well as to the incidence of LTC-IC
[25, 26]; these progenitors correspond
to the HPP-CFC1 of McNiece's classification.
The results obtained allowed the conclusion that our system
represents a simple, rapid and versatile method of estimating, within
a murine BMC population, a number of primitive and committed
progenitors, including largely quiescent stem cells. Studies of the
latter progenitors, in particular, would take advantage of the use of
this system, as it requires limited numbers of unenriched BMC and
short execution times, yet provides the possibility of evaluating the
progenitors' self-renewal and proliferative
potential.
|
ACKNOWLEDGMENT
|
|---|
The authors thank Prof. Massimo Olivotto, Department of
Experimental Pathology and Oncology, University of Florence, for moral
and material support to this work, and Dr. Elisabetta Rovida,
Department of Experimental Pathology and Oncology, for data analysis
and presentation. The project was funded by grants from Ministero
della Università e della Ricerca Scientifica e Tecnologica,
Consiglio Nazionale delle Ricerche, Associazione Italiana per la
Ricerca sul Cancro (AIRC) and Regione Toscana (Progetto
Qualità). Z.I. was the recipient of a fellowship from
AIRC. P.M. was supported by a grant from the Ministry of
Science and Technology of Serbia. V.P. was supported by a
grant from Conseil Regional du
Limousin.
|
References
|
|---|
-
Till JE, McCulloch EA. A direct
measurement of the radiation sensitivity of normal mouse bone marrow
cells. Radiat Res1961; 14
:213
-222.[Medline]
-
Schofield R, Dexter TM. Studies on the
self-renewal ability of CFU-S which have been serially transferred in
long-term culture or in vivo. Leuk Res
1985; 9
:305
-313.[Medline]
-
Hodgson GS, Bradley TR, Radley JM. The
organization of hemopoietic tissue as inferred from the effects of
5-fluorouracil. Exp Hematol
1982; 10
:26
-35.[Medline]
-
Van Zant G. Studies of hematopoietic stem
cells spared by 5-fluorouracil. J Exp Med
1984; 159
:679
-690.[Abstract/Free Full Text]
-
Dexter TM. Hematopoiesis in long-term
bone marrow cultures: a review. Acta Haematol
1979; 62
:299
-305.[Medline]
-
Chertkov JL, Deryigina EI, Drize NJ et
al. Individual clones of hemopoietic stem cells in murine long-term
bone marrow culture. Leukemia
1987; 1
:491
-496.[Medline]
-
van der Sluijs JP, De Jong JP, Brons NHC
et al. Marrow repopulating cells, but not CFU-S, establish long-term
in vitro hemopoiesis on a marrow-derived stromal layer. Exp
Hematol
1990; 18
:893
-896.[Medline]
-
Ploemacher RE, van der Sluijs JP, Voerman
JS et al. An in vitro limiting-dilution assay of long-term
repopulating hematopoietic stem cells in the
mouse. Blood
1989; 74
:2755
-2763.[Abstract/Free Full Text]
-
Ploemacher RE, van der Sluijs JP, van
Beurden CAJ et al. Use of limiting-dilution type long-term marrow
cultures in frequency analysis of marrow-repopulating and spleen
colony-forming hematopoietic stem cells in the
mouse. Blood
1991; 78
:2527
-2533.[Abstract/Free Full Text]
-
Quesenberry PJ. Whiteness of
Worchester. Exp Hematol
1998; 26
:455
-456
(editorial).
-
Bradley TR, Hodgson GS. Detection of
primitive macrophage progenitor cells in mouse bone
marrow. Blood
1979; 54
:1446
-1450.[Abstract/Free Full Text]
-
McNiece IK, Stewart FM, Deacon DM et
al. Detection of a human CFC with a high proliferation
potential. Blood
1989; 74
:609
-612.[Abstract/Free Full Text]
-
McNiece IK, Bradley TR, Kriegler AA et
al. Subpopulations of mouse bone marrow high-proliferative-potential
colony-forming cells. Exp Hematol
1986; 14
:856
-860.[Medline]
-
McNiece IK, Bertoncello I, Kriegler AB
et al. Colony-forming cells with high proliferative potential
(HPP-CFC). Int J Cell Cloning
1990; 8
:146
-160.[Abstract]
-
Lu L, Xiao M, Shen RN et
al. Enrichment, characterization, and responsiveness of single
primitive CD34+++ human umbilical blood hematopoietic
progenitors with high proliferative and replating
potential. Blood
1993; 81
:41
-48.[Abstract/Free Full Text]
-
Perez-Oteyza J, Ramos P, Testa N et
al. High-dose granulocyte-colony stimulating factor (G-CSF) in vitro
induces the growth of high proliferative potential colony forming
cells (HPP-CFC) in patients undergoing blood stem cell
mobilization. Exp Hematol
1997; 25
:516
-520.[Medline]
-
McNiece IK, Stewart FM, Deacon DM et
al. Synergistic interactions between hematopoietic growth factors as
detected by in vitro mouse bone marrow colony formation. Exp
Hematol
1988; 16
:383
-388.[Medline]
-
Milenkovic P, Ivanovic Z,
Stosic-Grujicic S. The in vivo effect of recombinant human
interleukin-1 receptor antagonist on spleen colony forming cells after
radiation-induced myelosuppression. Eur Cytokine Netw
1995; 6
:177
-180.[Medline]
-
Berardi A, Wang A, Levine J et
al. Functional isolation and characterization of human hematopoietic
stem cells. Science1995; 267
:104
-108.[Abstract/Free Full Text]
-
Hodgson GS, Bradley TR. Properties of
haematopoietic stem cells surviving 5-fluorouracil treatment: evidence
for a pre-CFU-S cell? Nature
1979; 281
:381
-382.[Medline]
-
Lord BJ, Woolford LB. Proliferation of
spleen colony-forming units (CFU-S8, CFU-S13) and cells with
marrow-repopulating ability. Stem Cells
1993
:11
:212
-217.[Abstract]
-
Bradley TR, Hodgson GS, Bertoncello
I. Characteristics of macrophage progenitor cells with high
proliferative potential: their relationships to cells with marrow
repopulating ability in 5FU-treated mouse bone marrow. In: Baum SJ,
Ledney GD, van Bekkum DW, eds. Experimental Hematology Today 1979. New
York: Karger 1980;285-297.
-
Jovcic G, Ivanovic Z,
Biljanovic-Paunovic L et al. The effect of IL-1 receptor antagonist on
the proliferation of hematopoietic progenitor cells in regenerating
bone marrow. Leukemia
1996; 10
:564
-569.[Medline]
-
Pragnell IB, Wright EG, Lorimore SA et
al. The effect of stem cell proliferation regulators demonstrated with
an in vitro assay. Blood
1988; 72
:196
-201.[Abstract/Free Full Text]
-
Watt SM, Visser JWM. Recent advances in
the growth and isolation of primitive human hemopoietic progenitor
cells. Cell Prolif
1992; 25
:263
-297.[Medline]
-
Boggs DR, Boggs SS, Saxe DF et
al. Hematopoietic stem cells with high proliferative potential. Assay
of their concentration in marrow by frequency and duration of cure of
W/Wv mice. J Clin Invest
1982; 70
:242
-253.
accepted for publication May 24, 1999.
This article has been cited by other articles:
|
|
|
|
 
F. R. Santoni de Sio, P. Cascio, A. Zingale, M. Gasparini, and L. Naldini
Proteasome activity restricts lentiviral gene transfer into hematopoietic stem cells and is down-regulated by cytokines that enhance transduction
Blood,
June 1, 2006;
107(11):
4257 - 4265.
[Abstract]
[Full Text]
[PDF]
|
|
|
Top
Abstract
Introduction
