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HEMATOPOIESIS
From the Stem Cell Laboratory, Institute of Laboratory
Medicine, University Hospital of Lund, Sweden.
Although long-term repopulating hematopoietic stem cells (HSC) can
self-renew and expand extensively in vivo, most efforts at expanding
HSC in vitro have proved unsuccessful and have frequently resulted in
compromised rather than improved HSC grafts. This has triggered the
search for the optimal combination of cytokines for HSC expansion.
Through such studies, c-kit ligand (KL), flt3 ligand (FL),
thrombopoietin, and IL-11 have emerged as likely positive regulators of
HSC self-renewal. In contrast, numerous studies have implicated a
unique and potent negative regulatory role of IL-3, suggesting perhaps
distinct regulation of HSC fate by different cytokines. However, the
interpretations of these findings are complicated by the fact that
different cytokines might target distinct subpopulations within the HSC
compartment and by the lack of evidence for HSC undergoing
self-renewal. Here, in the presence of KL+FL+megakaryocyte growth and
development factor (MGDF), which recruits virtually all
Lin In addition to their pluripotentiality, long-term
repopulating hematopoietic stem cells (HSC) have a unique ability to
undergo self-renewing cell divisions, as illustrated through their
expansion in vivo during ontogeny and after
transplantation.1-3
The self-renewing potential of HSC has stimulated extensive efforts at
mimicking this process in vitro to develop clinical applications, such
as retroviral-mediated stem cell gene therapy, and to expand the number
of stem cells to improve the quality of stem cell
transplants.3-7 It is clearly established that a minimum
number of long-term repopulating HSC (LTRC) is required to ensure
durable engraftment after transplantation,6 and LTRC may
be the best short-term repopulating cells.8,9
Retroviral marking studies in mice have supported that LTRC can undergo
self-renewal in vitro.10-13 However, with the exception of
one recent study,14 the objective of expanding ex vivo the number of murine LTRC remains elusive.3 Rather, most
previous studies have implicated that ex vivo culture of HSC results in compromised, or at best maintained, LTRC activity.3,15-19
Thus, the mechanisms governing HSC self-renewal remain to be established.
As the number of cloned cytokines promoting the growth of candidate
stem cells has increased, so has the focus on their potential role and
their use in stem cell expansion.6,7,20,21 The lack of a
net stem cell expansion under such conditions could be explained by an
inability of the cytokines to promote HSC divisions, and thus the
remaining stem cell activity could be derived from cytokine-unresponsive stem cells. In fact, findings from some studies
have supported this.17,22 Conversely, the finding in one
study of increased LTRC after ex vivo expansion14 would be
difficult to reconcile unless the repopulating HSC had undergone cell
divisions. In support of this, recent studies23 from our laboratory, in which murine HSC were successfully expanded or maintained in response to defined cytokines, demonstrated that most, if
not all, the reconstituting activity was retained in cells that
underwent multiple cell divisions. Significantly, these studies
unequivocally demonstrated that combinations of early-acting cytokines
efficiently promote LTRC to undergo self-renewing divisions.
The heterogeneous expression of various cytokine receptors on HSC could
explain why some cytokines might be more efficient than others at
promoting the recruitment of HSC into proliferation,24 a
requisite for HSC expansion. More intriguingly, some studies have
suggested that certain cytokines, in particular IL-3, may have a
negative impact on in vitro stem cell expansion in
mice16,19,25-27 and in humans.28,29 If this
effect is mediated by direct action on HSC, it could implicate a novel
negative regulation of stem cell self-renewal by IL-3, as previously
proposed.16,27 Evidence for this would be of great
importance because the role of cytokines in regulating the fate of HSC
is controversial30,31 and because most data support a
permissive rather than an instructive role of
cytokines.32-35 However, an important limitation to the
studies suggesting that IL-3 negatively affects long-term repopulating HSC is the lack of evidence for HSC self-renewal under the conditions used.22 In addition to potentially inducing commitment or
maturation rather than self-renewal, there are multiple alternative
mechanisms that may explain why IL-3 negatively affects stem cell
expansion. One is inhibition of the number of LTRC recruited into
proliferation. Although they usually promote growth, colony-stimulating
factors have, under special circumstances, been shown to suppress
growth.20 Another alternative mechanism concerns the
unique effects on the cell cycle distribution of HSC because
reconstituting stem cells have been proposed to reside predominantly in
G0.3,17,36 A third alternative mechanism is
that the negative effects of IL-3 may not be directly mediated on the
HSC but may be mediated through effects on other cells in culture,
resulting in the activation of negative regulators and pathways. In
this regard, it is noteworthy that the ex vivo expansion studies that
have yielded the most promising results have used purified target
cells.14,15,19,37-40,41
To better dissect the mechanisms governing the negative
influence of IL-3 on in vitro HSC expansion, we used a system in which we recently demonstrated that c-kit ligand (KL), flt3-ligand (FL), and
megakaryocyte growth and development factor (MGDF) efficiently promote
proliferation of murine
Lin Hematopoietic growth factors
Purification of
Lin Single-cell cultures LSK cells or LSKCD34 cells (120 cells/group) were
seeded in Terasaki plates (Nunc, Kamstrup, Denmark) at a density of
0.25 cell/well (LSKCD34) or 1 cell/well (LSK) in 20 µL
X-Vivo 15 (BioWhittaker, Walkersville, MD) supplemented with 1%
detoxified bovine serum albumin (BSA; StemCell Technologies, Vancouver,
Canada) as previously reported.47 Wells were scored for
cell growth after 10 to 12 days of culture at 37°C. Because the
statistical probability of a well (based on Poisson distribution) not
receiving a cell is 37%, when plated at a density of 1 cell/well, the
theoretical maximum number of expected clones was 76 for each group. In
some experiments, each well was inspected within 12 hours of plating,
and only wells containing 1 cell were included in the experiment,
giving similar results.
Ex vivo expansion cultures Ex vivo expansion cultures were performed using either serum-free (X-Vivo 15 with 1% BSA; BioWhittaker) or serum-containing medium (IMDM supplemented with 20% fetal calf serum [FCS]; BioWhittaker). Cell densities were never allowed to exceed 106 cells/mL. This was possible by seeding cells at low numbers (300 cells/mL) and adding prewarmed fresh medium and cytokines on day 7 of culture. At the time of harvest, cells were recovered, and each flask/well was washed twice with phosphate-buffered saline (PBS) without Ca/Mg (BioWhittaker) and cell dissociation buffer (Life Technologies, Gaithersburg, MD) to recover additional adherent cells. Cells were then washed and diluted in serum-free media together with competitor cells to make a final injection volume of 0.5 mL/mouse.High-resolution cell division tracking of candidate murine stem cells Staining and flow cytometric procedures for cell division tracking have been described previously.23,49 Briefly, 5- and 6-carboxyfluorescein diacetate succinimidyl ester (CFSE; Molecular Probes, Eugene, OR) from a 5 mmol/L stock in dimethyl sulfoxide was added to cells (at 5 × 106 cells/mL) in PBS to give a final concentration of 1 µmol/L. Cells were incubated at 37°C for 10 minutes, and the staining was stopped by adding a 10-fold excess of PBS with 10% to 20% FCS, after which cells were washed twice. To allow unbound dye to diffuse from cells, labeled cells were incubated for 12 to 15 hours at 37°C in serum-free media supplemented with KL (50 ng/mL) to promote stem cell survival rather than proliferation. Cells were subsequently stained with rat antimouse Sca-1 phycoerythrin and anti-c-kit APC and were sorted based on the dual expression of Sca-1 and c-kit from a 40- to 50-channel-wide sort gate set around the mean fluorescent channel for CFSE. Sorted cells were used in expansion cultures with indicated cytokines and, starting at 20 hours, were analyzed for "proliferation history" on a FACSCalibur (Becton Dickinson) at 12-hour intervals until 140 hours using identical instrument settings. As a control for undivided cells, a sample of freshly sorted cells was fixated in 2% paraformaldehyde (Sigma). We noted that some (though limited) CFSE diffusion appeared after the 12- to 15-hour preincubation (Bryder D, Jacobsen SEW, unpublished observation, 1998). However, because this diffusion of CFSE was limited and was never close to one cell division within a 12-hour interval, it was possible to determine accurately the number of cell divisions by overlaying histograms from each analysis time point.In vivo reconstitution experiments Animal experiments were conducted after approval was obtained from the local ethics committee. Ly5.1+ LSK or LSK CD34 cells were used as donor cells. Eight- to
12-week-old C57bl/6 mice (Ly5.2) were transplanted with freshly
isolated HSC or with the expansion equivalent of the same number of
HSC, together with Ly5.2+ competitor cells (150 000 or
200 000 unfractionated BM cells). This was done in part to allow the
quantification of reconstitution activity and in part to ensure the
survival of mice transplanted with potentially compromised grafts. Four
to 8 hours before transplantation, each mouse received a lethal dose of
irradiation of 950 cGy using a cesium Cs 137 source (Instrument AB
Scanditronix; Husbyborg, Uppsala, Sweden). All mice were kept in
individually ventilated cages throughout the experiments and given
sterile food and autoclaved acidified water.
Peripheral blood was drawn from transplanted mice from the
retro-orbital sinus and analyzed at 6 weeks and at 4 months after transplantation for donor (Ly5.1+) and for
competitor/recipient-derived (Ly5.2+) reconstitution by
flow cytometry. Lineage distribution was examined using antibodies
against CD3 For secondary transplantations, a half-femur equivalent (representing approximately 5% of the total BM) from primary recipients was injected into each lethally irradiated secondary recipient. Peripheral blood from secondary transplanted mice were analyzed 3 to 4 months after transplantations, as described above. Statistics The Mann-Whitney U test was used throughout to determine the statistical significance between the treatment groups.
We recently demonstrated that ex vivo culture of LSK cells in serum-free medium in response to KL+FL+MGDF (KFM) efficiently promotes stem cell proliferation while sustaining long-term reconstituting activity.23 Preliminary studies in our laboratory suggested that IL-11, which, in contrast to IL-3, has been thought to affect stem cell expansion positively,2,14,19,42-44 had effects similar to those of IL-3 on the KFM-induced proliferation of LSK cells. If this is so, this could provide a unique system for determining how 2 cytokines with comparable effects on stem cell proliferation might have opposing effects on stem cell fate. The kinetics and extent of cellular expansion of LSK cells incubated in
KFM3 and KFM11 were indistinguishable (Figure
1). Whereas KFM resulted in a 460-fold
cellular expansion at 240 hours, as much as 6300- and 6400-fold cell
expansion was observed with the addition of IL-3 and IL-11,
respectively. The addition of IL-3 or IL-11 did not alter the number of
emerging LSK clones (all combinations tested resulted in the
recruitment of virtually all LSK cells), though both factors enhanced
the average clone size (Table 1).
Significantly, this finding would imply that any potential differences
observed in the abilities of KFM, KFM3, and KFM11 to maintain or expand
LTRC would probably not be explained by the recruitment of different
stem cell populations into proliferation.
Because HSC may undergo only a limited number of self-renewal divisions
and have been demonstrated to undergo asymmetric and asynchronous cell
divisions during in vitro culture,25,50,51 it may be
possible to explain differences between cytokines in promoting ex vivo
stem cell expansion by the distinctions in the number of induced stem
cell divisions. To address this specifically, LSK cells were stained
with CFSE to allow high-resolution tracking of the number of cell
divisions undergone in response to KFM, KFM3, and KFM11 (Figure
2). Both IL-3 and IL-11 increased the average number of KFM-stimulated cell divisions after 44 hours culture,
demonstrating that each cytokine acts at an early stage to enhance the
proliferative activity of LSK cells. Furthermore, CFSE profiles
(performed at 12-hour intervals) demonstrated indistinguishable kinetics for KFM3- and KFM11-induced cell division. Of particular relevance for the subsequent LTRC ex vivo expansion experiments, virtually all LSK cells underwent at least one cell division by 68 hours in response to KFM3 and KFM11, whereas no cells underwent fewer
than 5 divisions after 140 hours of culture (Figure 2). Thus, KFM3 and
KFM11 reveal an identical and efficient pattern of promoting LSK
recruitment and proliferation in vitro.
We next investigated the ability of KFM, in the absence or presence of
IL-3 or IL-11, to expand the in vivo multilineage reconstituting activity of LSK cells. Because the extent or length of ex vivo culture
could affect the level of HSC activity recovered,14 we
specifically investigated reconstitution activity after 56 to 80 hours,
104 to 116 hours, and 240 hours of culture in serum-free medium. At
each time point, the total cellular expansion was established (Figure
1). Cells cultured in all 3 cytokine combinations for 56 to 80 hours
and for 104 to 116 hours had similar and sustained multilineage
reconstituting activity 6 weeks after transplantation (when compared to
uncultured cells; Figure 3A). In
contrast, LSK cells expanded for as much as 10 days had enhanced
reconstituting activity (2.4- to 2.7-fold compared to fresh cells;
P < .001 for all combinations), but there was no clear
difference observed between the 3 cytokine combinations. After 4 months, a similar pattern of reconstituting activity was observed
(Figure 3B). When compared to uncultured input cells, cells expanded in
all 3 cytokine combinations for fewer than 116 hours showed sustained
reconstituting activity, whereas 240 hours of expansion resulted in the
following increases in reconstituting activity: KFM, 2.5-fold
(P < .01); KFM3, 2.2-fold (P < .001);
KFM11, 1.4-fold (not statistically significant). Of importance,
transplanted cells from the various expansion conditions showed similar
contributions to B, T, and myeloid blood cells as uncultured cells
(Table 2). Unexpectedly, we did not
observe any negative effects of IL-3 (or positive effects of IL-11) on
either short- or long-term reconstituting activity or lineage
contribution of KFM-expanded LSK cells. Rather, the extended culture of
LSK cells exposed to IL-3, in which total cell numbers had been
increased by as much as 6400-fold, showed enhanced reconstituting
activity.
Because serial transplantations put enhanced proliferative pressure on
HSC,52-54 the ability to reconstitute secondary recipients is considered a more stringent measure of true LTRC activity. Thus, to
demonstrate unequivocally that IL-3 did not negatively affect the
long-term reconstituting activity of LSK cells and that prolonged
expansion culture did not exhaust the stem cell pool, BM cells from
primary recipients of KFM and KFM3-expanded cells were transplanted
into secondary recipients. After another 4 months, a much higher
secondary reconstituting activity of KFM and KFM3-expanded cells was
observed than was observed of the same number of uncultured input cells
(Figure 4).
Because it has been suggested that LSKCD34+ and
LSKCD34
Our findings of enhanced LTRC activity after extensive and prolonged
HSC proliferation in the presence of IL-3 was a surprise in light of
previous studies of murine and human stem cells that implicated IL-3
might have a potent negative effect on in vitro stem cell
self-renewal.16,19,25,26,28,29 Although there were many
possible explanations for this finding (addressed in more detail in the
"Discussion"), most ex vivo expansion studies of murine stem cells
showing a loss of reconstituting activity had been performed in
FCS-containing medium.16-18,56 The only previous studies
showing in vitro expansion of murine LTRC activity (in the absence of
IL-3) were, as were our studies, performed under serum-free
conditions.14 Although no differences in the recruitment
of single LSK cells into proliferation were observed in serum-free and
FCS-containing medium (S. E. W. Jacobsen and D. Bryder,
unpublished observations) and the levels of total cell expansion were
comparable (slightly higher in FCS-containing medium; Figure
5A), the effect on the preservation of in
vivo reconstituting activity was strikingly different. Whereas LSK
cells that expanded for 116 or 240 hours in the presence of KFM3 or
KFM11 under serum-free conditions demonstrated sustained or enhanced
short-term (6 weeks; Figure 5B) or long-term (4 months; Figure 5C)
reconstituting activity, cells cultured in FCS-containing medium always
had severely reduced reconstituting activity, regardless of cytokine
combination or duration of culture. However, at 240 hours, a clear
tendency was observed toward more pronounced reduction in
reconstitution activity in IL-3- than in IL-11-containing cultures
(P < .01).
Because the serum-free and FCS-containing media differed not only with
regard to the absence or presence of FCS (see "Materials and
methods"), a final experiment was designed to address more specifically the effects of FCS. The fact that dramatically reduced LTRC activity of LSK cells was observed not only after culture in the
standard (IMDM-based) FCS medium but also when FCS was added to the
serum-free medium (Table 4) clearly
supported a detrimental effect of the FCS on stem cell ex vivo
expansion in the current studies.
Because we could not exclude that the negative effects of IL-3 on LTRC activity were dependent on the specific interacting cytokines, we also examined the effect of KL+IL6+IL11+EPO+IL-3 on LSK ex vivo expansion (Table 4). This specific cytokine combination has previously (in FCS-containing cultures) been demonstrated to have a severe negative effect on maintenance of LTRC.16,19 In striking contrast to the FCS-containing culture that reproduced this negative effect, the LTRC activity of cells exposed to the same cytokines in SF medium was enhanced in comparison with uncultured control cells (Table 4).
Although much progress has been made in recent years toward the development of better surrogate human stem cell assays,57-61 it remains unclear to what degree these assays detect true long-term repopulating HSC activity. Thus, clinical stem cell and gene-marking protocols will eventually have to address whether the recent success in expanding and retroviral-transducing candidate human HSC translates into true LTRC activity.37,40,41,62 In the meantime, complimentary studies in animal models that provide true assays for LTRC are paramount for a better understanding of the processes regulating HSC fate. The HSC in mouse remain phenotypically and functionally the best characterized,3,48,63 and ample evidence suggest that human HSC are subjected to the same regulatory networks. This appears particularly evident with regard to the potential role of various cytokines in regulating HSC proliferation and differentiation.20,21 Thus, studies on murine HSC continue to govern efforts at using cytokines to promote the expansion of human HSC for use in stem cell transplantation and gene therapy.4-7 However, the success in expanding murine HSC with sustained long-term repopulating ability has been variable and limited and has more frequently resulted in compromised rather than enhanced LTRC activity.3,15-19 Although a number of variables can explain the lack of success in expanding LTRC, major focus has been directed to the ability of different cytokines (and combinations of these) to promote stem cell expansion.6,7 Whereas cytokines appear to support the differentiation of hematopoietic cells primarily through permissive rather than instructive mechanisms,32-35 their role in regulating stem cell fate (self-renewal, differentiation, or apoptosis) remains unclear.3,30,31 Some studies have suggested that IL-11, in combination with FL, KL, or both, can positively affect ex vivo LTRC maintenance and expansion. 2,14,19,42-44 Others have revealed a severe negative effect of IL-3.16,19,25-27 These may imply distinct roles for these 2 cytokines in regulating stem cell fate. However, because HSC are also heterogeneous with regard to their cytokine receptor expression,24 it is difficult to exclude that differences observed between cytokine combinations might simply reflect heterogeneity in receptor expression and, thus, target cell populations. To overcome this limitation, we used a combination of KL, FL, and MGDF. We recently demonstrated23 that this combination efficiently recruits virtually all murine LSK HSC into proliferation while it maintains their long-term reconstituting activity. This, combined with the finding that IL-3 and IL-11 had a potent and indistinguishable ability to enhance the cycling of KFM-recruited LSK cells, allowed us to compare their effects on self-renewal divisions of the same population of HSC. Our finding that both IL-3 and IL-11 supported KFM-stimulated HSC self-renewal was surprising in light of previous studies suggesting opposing effects of these 2 cytokines in this process.16,19,25 As illustrated through the increase in total cellular expansion and the high-resolution cell division tracking, IL-3 clearly enhanced the cycling of LSK HSC without negatively affecting self-renewal after as much as 10 days of incubation. Although it remains possible that IL-3 may only negatively affect HSC self-renewal induced by certain cytokine combinations, this seems unlikely because IL-3 did not abrogate HSC self-renewal divisions induced in response to KL+IL-6+IL-11+EPO, for which a negative effect of IL-3 has been demonstrated.16,19 Furthermore, though IL-3 also has been demonstrated to regulate negatively the lymphoid potential of HSC,64,65 IL-3 (and IL-11) did not affect the relative levels of myeloid and lymphoid reconstitution by expanded stem cells. Although these studies clearly demonstrate that IL-3 can support multiple self-renewing divisions of LTRC, they only provide clues as to why other studies have demonstrated that IL-3 can have a severe negative effect on ex vivo expansion of murine HSC.16,19,25-27 Common to all studies showing the suppression of murine LTRC activity by IL-3 is the presence of FCS in the expansion cultures, which, in the current study and in other recent studies in humans, proved detrimental for HSC expansion.23,66 Although the specific FCS (and concentration) used might vary in its ability to support HSC, as illustrated through a few studies demonstrating maintenance of LTRC activity,19,26 these studies clearly demonstrate that the presence of FCS can be detrimental to the preservation of HSC during ex vivo expansion. The mechanism by which FCS abrogated stem cell maintenance was not addressed in the current study. However, because the level of recruitment and total cellular expansion of LSK cells in response to KFM3 and KFM11 were comparable in the serum-free and FCS-containing medium, the negative effect of FCS is unlikely to result from an effect on HSC proliferation. However, we have observed that FCS-containing cultures are more efficient at supporting the generation of fully differentiated myeloid (granulocyte-macrophage) cells (Bryder D, Jacobsen SEW, unpublished observations). In that regard, we did, in the presence of FCS, observe a facilitating effect of IL-3 on HSC loss in culture at 116 hours and at 240 hours. In contrast, in the presence of IL-11, the levels of HSC recovered slightly after 240 hours, which was similar to what we observed in serum-free media. Because the direct effects of IL-3 and IL-11 on proliferation of LTRC seem comparable, the possibility that the negative effects of IL-3 on HSC self-renewal might be mediated indirectly through effects on mature cells, generated in cultures, should be entertained. Studies of primitive human progenitors have suggested that a negative effect of IL-3 may be dependent on relative cytokine concentrations.28 Although we cannot exclude such an effect on murine HSC, the levels of IL-3 and other cytokines used in the current study were comparable to those used in studies showing negative effects of IL-3.26 The proliferation history of LSK cells, as established through CFSE
staining, clearly demonstrated that all LTRC had undergone multiple (at
least 5) cell divisions after 140 hours of culture in KFM3 or KFM11,
unequivocally demonstrating that LTRC can undergo multiple
self-renewing divisions in vitro. This conclusion was also
supported by the studies on single LSKCD34 Although repopulating activity was enhanced in IL-3- and IL-11-containing cultures, specific quantification of expanded LTRC was not performed to demonstrate unequivocally an increase in stem cell numbers. However, the expanded HSC displayed enhanced long-term reconstituting activity, not only in primary but also in secondary lethally irradiated recipients, with lineage distribution indistinguishable from that of freshly isolated cells. Furthermore, the finding of mice lacking both myeloid and lymphoid reconstitution when transplanted with fresh LSKCD34-cells, but not expanded cells, supports an expansion in LTRC numbers. That ex vivo expansion for 10 days was associated with enhanced reconstituting activity suggests that some of the self-renewing divisions must have been symmetric. However, the fact that a 2- to 3-fold expansion in LTRC activity was accompanied by at least 5 cell divisions by all LTRC and by as much as 400- to 6500-fold total cellular expansion implies either that most self-renewing divisions are asymmetric (resulting in the generation of only one new HSC) or that a high frequency of HSC, generated through symmetric cell divisions, undergoes apoptosis or is otherwise compromised in its ability to engraft. One of the most intriguing findings in our studies was that the magnitude of HSC expansion was higher after 240 hours than at 116 hours, though all HSC appeared to have divided multiple times by 116 hours. The explanation for this remains unclear, but one possibility is that increased culture time enhances the likelihood of symmetric self-renewal divisions. Alternatively, our culture conditions might only support the preservation and expansion of a subset of HSC, resulting in loss of some HSC during the first days of culture. A better understanding of the processes regulating stem cell fate will prove crucial for the development of a more efficient expansion of long-term repopulating stem cells. Regardless, the current study and that of Miller et al14 support the feasibility of prolonged expansion of HSC to promote retroviral-mediated gene transfer or to purge autologous stem cell transplants while maintaining the long-term repopulating ability of the graft.4-7
Submitted February 17, 2000; accepted May 2, 2000.
Supported by grants from ALF (Government Public Health Grant); Berta Kamprad Foundation; Crafoord Foundation; Georg Danielsson Foundation; Gunnar, Arvid and Elisabeth Nilsson Foundation; Harald and Greta Jeansson's Foundation; Thelma Zoega's Foundation; John and Augusta Persson Foundation; Medical Faculty, University of Lund; Swedish Medical Research Council (MFR); Svensson Siblings Foundation; O and E and Edla Johansson Foundation; Royal Physiographic Society in Lund; Swedish Foundation for Strategic Research; Swedish Cancer Society; Swedish Society of Pediatric Cancer; and Tobias Foundation.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: David Bryder, Stem Cell Laboratory, Institute of Laboratory Medicine, University Hospital of Lund, 221 85 Lund, Sweden; e-mail: David.Bryder{at}molmed.lu.se.
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  S. D. Yogesha, S. M. Khapli, R. K. Srivastava, L. S. Mangashetti, S. T. Pote, G. C. Mishra, and M. R. Wani IL-3 Inhibits TNF-{alpha}-Induced Bone Resorption and Prevents Inflammatory Arthritis J. Immunol., January 1, 2009; 182(1): 361 - 370. [Abstract] [Full Text] [PDF]
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L. A. Thoren, K. Liuba, D. Bryder, J. M. Nygren, C. T. Jensen, H. Qian, J. Antonchuk, and S.-E. W. Jacobsen Kit Regulates Maintenance of Quiescent Hematopoietic Stem Cells J. Immunol., February 15, 2008; 180(4): 2045 - 2053. [Abstract] [Full Text] [PDF]
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 M. Svensson, J. Marsal, H. Uronen-Hansson, M. Cheng, W. Jenkinson, C. Cilio, S. E. W. Jacobsen, E. Sitnicka, G. Anderson, and W. W. Agace Involvement of CCR9 at multiple stages of adult T lymphopoiesis J. Leukoc. Biol., January 1, 2008; 83(1): 156 - 164. [Abstract] [Full Text] [PDF]
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 N. Buza-Vidas, M. Cheng, S. Duarte, H. Nozad, S. E. W. Jacobsen, and E. Sitnicka Crucial role of FLT3 ligand in immune reconstitution after bone marrow transplantation and high-dose chemotherapy Blood, July 1, 2007; 110(1): 424 - 432. [Abstract] [Full Text] [PDF]
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M. Magnusson, A. C. M. Brun, N. Miyake, J. Larsson, M. Ehinger, J. M. Bjornsson, A. Wutz, M. Sigvardsson, and S. Karlsson HOXA10 is a critical regulator for hematopoietic stem cells and erythroid/megakaryocyte development Blood, May 1, 2007; 109(9): 3687 - 3696. [Abstract] [Full Text] [PDF]
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J. M. Nygren, D. Bryder, and S. E. W. Jacobsen Prolonged Cell Cycle Transit Is a Defining and Developmentally Conserved Hemopoietic Stem Cell Property J. Immunol., July 1, 2006; 177(1): 201 - 208. [Abstract] [Full Text] [PDF]
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H. Qian, K. Tryggvason, S. E. Jacobsen, and M. Ekblom Contribution of {alpha}6 integrins to hematopoietic stem and progenitor cell homing to bone marrow and collaboration with {alpha}4 integrins Blood, May 1, 2006; 107(9): 3503 - 3510. [Abstract] [Full Text] [PDF]
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E. F. Srour, X. Tong, K. W. Sung, P. A. Plett, S. Rice, J. Daggy, C. T. Yiannoutsos, R. Abonour, and C. M. Orschell Modulation of in vitro proliferation kinetics and primitive hematopoietic potential of individual human CD34+CD38-/lo cells in G0 Blood, April 15, 2005; 105(8): 3109 - 3116. [Abstract] [Full Text] [PDF]
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L. Yang, D. Bryder, J. Adolfsson, J. Nygren, R. Mansson, M. Sigvardsson, and S. E. W. Jacobsen Identification of Lin-Sca1+kit+CD34+Flt3- short-term hematopoietic stem cells capable of rapidly reconstituting and rescuing myeloablated transplant recipients Blood, April 1, 2005; 105(7): 2717 - 2723. [Abstract] [Full Text] [PDF]
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 L. De Felice, C. Tatarelli, M. G. Mascolo, C. Gregorj, F. Agostini, R. Fiorini, V. Gelmetti, S. Pascale, F. Padula, M. T. Petrucci, et al. Histone Deacetylase Inhibitor Valproic Acid Enhances the Cytokine-Induced Expansion of Human Hematopoietic Stem Cells Cancer Res., February 15, 2005; 65(4): 1505 - 1513. [Abstract] [Full Text] [PDF]
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A. M. Hanash and R. B. Levy Donor CD4+CD25+ T cells promote engraftment and tolerance following MHC-mismatched hematopoietic cell transplantation Blood, February 15, 2005; 105(4): 1828 - 1836. [Abstract] [Full Text] [PDF]
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L. Yang, I. Dybedal, D. Bryder, L. Nilsson, E. Sitnicka, Y. Sasaki, and S. E. W. Jacobsen IFN-{gamma} Negatively Modulates Self-Renewal of Repopulating Human Hemopoietic Stem Cells J. Immunol., January 15, 2005; 174(2): 752 - 757. [Abstract] [Full Text] [PDF]
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J. Antonchuk, C. D. Hyland, D. J. Hilton, and W. S. Alexander Synergistic effects on erythropoiesis, thrombopoiesis, and stem cell competitiveness in mice deficient in thrombopoietin and steel factor receptors Blood, September 1, 2004; 104(5): 1306 - 1313. [Abstract] [Full Text] [PDF]
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Y. Sasaki, C. T. Jensen, S. Karlsson, and S. E. W. Jacobsen Enforced expression of cyclin D2 enhances the proliferative potential of myeloid progenitors, accelerates in vivo myeloid reconstitution, and promotes rescue of mice from lethal myeloablation Blood, August 15, 2004; 104(4): 986 - 992. [Abstract] [Full Text] [PDF]
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D. Bryder, Y. Sasaki, O. J. Borge, and S.-E. W. Jacobsen Deceptive Multilineage Reconstitution Analysis of Mice Transplanted with Hemopoietic Stem Cells, and Implications for Assessment of Stem Cell Numbers and Lineage Potentials J. Immunol., February 1, 2004; 172(3): 1548 - 1552. [Abstract] [Full Text] [PDF]
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I. Dybedal, L. Yang, D. Bryder, I. Aastrand-Grundstrom, K. Leandersson, and S. E. W. Jacobsen Human reconstituting hematopoietic stem cells up-regulate Fas expression upon active cell cycling but remain resistant to Fas-induced suppression Blood, July 1, 2003; 102(1): 118 - 126. [Abstract] [Full Text] [PDF]
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 D. Bryder, V. Ramsfjell, I. Dybedal, K. Theilgaard-Monch, C.-M. Hogerkorp, J. Adolfsson, O. J. Borge, and S. E. W. Jacobsen Self-Renewal of Multipotent Long-Term Repopulating Hematopoietic Stem Cells Is Negatively Regulated by FAS and Tumor Necrosis Factor Receptor Activation J. Exp. Med., October 1, 2001; 194(7): 941 - 952. [Abstract] [Full Text] [PDF]
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I. Dybedal, D. Bryder, A. Fossum, L. S. Rusten, and S. E. W. Jacobsen Tumor necrosis factor (TNF)-mediated activation of the p55 TNF receptor negatively regulates maintenance of cycling reconstituting human hematopoietic stem cells Blood, September 15, 2001; 98(6): 1782 - 1791. [Abstract] [Full Text] [PDF]
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J. Domen and I. L. Weissman Hematopoietic Stem Cells Need Two Signals to Prevent Apoptosis; Bcl-2 Can Provide One of These, Kitl/C-KIT Signaling the Other J. Exp. Med., December 18, 2000; 192(12): 1707 - 1718. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
(T cells), B220 (B cells), and a combination of
Mac-1/Gr-1 (myeloid cells) along with antibodies against Ly5.1 and
Ly5.2 (all from PharMingen).
) bone marrow long-term culture-initiating cells (LTC-IC), extended LTC-IC, and murine in vivo long-term reconstituting stem cells.
Blood.
1999;94:4093-4102