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HEMATOPOIESIS
From INSERM U 362, Institut Gustave Roussy, Villejuif,
and INSERM U 474, Hôpital Port Royal, Paris, France.
Stem cell proliferation induced by potent cytokines usually leads
to a loss of primitive potential through differentiation. In this
study, the ability of cytokines and murine MS5 stromal cells to
independently regulate the proliferation and long-term culture-initiating cell (LTC-IC) activity of primitive
CD34+CD38low/neg human bone marrow cells was
evaluated. To compare populations with identical proliferation
histories, cells were labeled with carboxy fluorescein diacetate
succinimidyl ester, and LTC-IC activity was assessed 4 days later in
cells that had accomplished the same number of divisions with or
without MS5 cells. MS5 cells counteracted dramatically the loss of
LTC-IC activity observed in the presence of cytokines alone. Thus, in
the presence of MS5 cells, means of 1233 (n = 5) and 355 (n = 9)
LTC-IC-derived colony-forming cells (CFCs) were generated by 1000 cells that performed 3 and 4 divisions respectively, whereas 311 (n = 5) and 64 (n = 5) CFCs were generated by 1000 cells cultured
without MS5 cells. Interestingly, MS5 cells had no detectable effect on
the LTC-IC activity of cells that divided only twice in 4 days The loss of stem cell activity that invariably
occurs when primitive cells are induced to divide by cytokines
compromises attempts to obtain a net increase in the number of stem
cells for therapeutic purposes in transplantation or gene transfer with murine retroviral vectors. What can be demonstrated is an amplification of cells retaining similar functional properties, such as the ability
to reconstitute long-term culture (LTC) in vitro1 or nonobese diabetic-severe combined immunodeficient (NOD-SCID)
recipients in vivo.2,3 However, this is by no means proof
of self-renewal because the molecular identities of daughter and parent
cells cannot be demonstrated in the absence of known genetic mechanisms that control self-renewal versus differentiation. Thus, net increases in the number of long-term culture-initiating cell, (LTC-IC), human
NOD-SCID-reconstituting cells (rc), and, more rarely, murine long-term
repopulating cells4 have been described after a short-term exposure to cytokines, but usually the expansion does not exceed a
factor of 10, and the low frequency of proliferating cells
participating in the expansion of the stem cell compartment also
complicates evaluation of quantitative analyses of these responses.
Although stromal cells have been assigned contradictory functions, they
have often been viewed as important for the maintenance of primitive
progenitor activity,5 particularly in the lymphoid lineages.6 Coculture with stromal cells is the basis of
the LTC system. However, now that many purified cytokines are
available, several groups have demonstrated an additional ability of
stromal feeders to maintain primitive hematopoietic cell function in
cultures used for expansion7-9 or gene transfer
protocols.10 The contribution of cytokines, adhesion
molecules,11 and other stroma-derived regulators, such as
Notch ligands,12 in these effects has been reported,
though it is unclear whether stromal cells act by affecting cell
survival13 or by promoting or delaying cell-cycle
entry.14 One helpful strategy to monitor variations in
cell proliferation in these conditions has been to label the cells with
fluorescent dyes such as PKH-2, PKH-26,15 or more recently
carboxy fluorescein diacetate succinimidyl ester
(CFSE).16-19 Because these dyes are diluted by cell
division, they can be used to track the proliferative behavior of cells
in vitro and are thus useful to correlate changes in cell function with
successive cell divisions. This approach has been used recently to
identify cells that remain quiescent for long periods in vitro, even
when they are exposed to suboptimal concentrations of cytokines and
stromal feeders.20,21 It has also allowed cell
proliferation to be correlated with retention of primitive phenotypes
and functions (cobblestone area-forming cells and NOD-SCID
repopulation) by primitive progenitors exposed to cytokines for a short
period.18,19 These data have confirmed that actively
proliferating cells can retain LTC-IC or NOD-SCID-rc functions, but
only transiently.
Others and we22-25 have reported that murine stromal
cells can counteract the loss of stem cell activity of human bone
marrow CD34+CD38low/neg samples exposed to high
concentrations of cytokines. However, it remains unresolved whether
this effect of stromal cells resulted from the rate of proliferation of
an unidentified subpopulation of primitive cells. To address this
question, we labeled adult bone marrow-derived
CD34+CD38low/neg with CFSE to monitor
simultaneously cell divisions and concomitant changes in the LTC-IC
activity of cells exposed to high concentrations of cytokines in the
presence of murine MS5 stromal cells. The presence of MS5 cells caused
marked retention of a high LTC-IC potential by cells that completed 3 or 4 divisions in 4 days in comparison to cells cultured with cytokines
alone, without feeders, that showed a rapid loss of LTC-IC function.
These data demonstrate that the proliferation and differentiation of
primitive human hematopoietic cells can be independently regulated by
stroma-derived molecules.
Cell preparation and labeling
Short-term cultures
In 2 experiments, to increase the homogeneity of the input cells,
we performed double sorting, as illustrated in Figure
1. On day 4, we first selected cells that
had divided twice (slow-dividing cells), an aliquot of which was kept
to assay LTC-IC activity, with the remaining cells cultured 2 days
later with or without MS5 cells. At the end of the second culture
period, cells that had performed 2 more divisions (a total of 4 from
day 0) were again sorted, and their LTC-IC potentials were analyzed.
In one experiment, we sorted cells that had completed 4 divisions (4-division cells) and those that had completed fewer than 4 divisions (0- to 3-division cells) after 4 days in culture. Four-division cells were assayed in LTC, whereas 0- to 3-division cells were cultured for another 24 hours with and without MS5 cells. The next day (day 5), we again sorted separately 4-division cells and 0- to 3-division cells, and both fractions were handled as described previously. This step was repeated again after 6 and 7 days in culture. Statistical analysis Statistical significance was determined by Student t-test (paired data analysis).
Murine MS5 stromal cells do not alter the initial rate of division of CD34+CD38low/neg cells in short-term culture To investigate whether MS5 cells altered the function of CD34+CD38low/neg cells exposed to cytokines through a change in their proliferation rate, we labeled cells with CFSE16 and measured the LTC-IC activity of cells that underwent the same number of divisions in 4-day cytokine-supplemented cultures with or without MS5 cells. At the end of the 4 days, the nucleated cells were distributed in different peaks of fluorescence, each corresponding to one cell generation, as shown by FACS analysis (Figure 1, step 1). As would be expected from an equal partitioning of the dye between daughter cells at each division,28 there was a linear decrease in fluorescence from one peak to the next (not shown). The 0-division point was defined from the amount of fluorescence measured in input cells cultured with Colcemid.Our first observation was the heterogeneous rate of proliferation of
individual CD34+CD38low/neg cells; 6 or 7 generations of daughter cells could be identified independent of the
presence of MS5 cells (Figure 2). During
the first 4 days, more than 98% of
CD34+CD38low/neg cells exposed to the 6 cytokines had divided at least once and 60% at least 3 times, which
fits with the 5- to 10-fold overall increase observed in the total
number of nucleated cells. As shown in Figure 2, in the absence of MS5
cells, 81% ± 2% of the cells underwent 3 divisions or more, and
88% ± 3% underwent them in the presence of MS5 (mean ± SEM of 5 experiments). There was also no significant difference in
the proportions of cells in the different CFSE peaks in MS5
cell-containing or MS5-free cultures, which further shows that the MS5
feeders had no effect on cell proliferation rate. A small population
remained quiescent or divided only once, and, interestingly, this
fraction was reproducibly lower in the presence of MS5 cells (0.5%)
than in their absence (3.3%). Consequently, the proportion of cells
that had accomplished more than 3 divisions appeared slightly higher on
stromal cells (63% versus 50% without MS5 cells), though these latter
differences did not reach significance.
Thus, the induction of cell division in CD34+CD38low/neg cells was primarily controlled by cytokines. This agrees with our previous results showing that few CD34+CD38low/neg cells with LTC-IC function, cultured as described above, survived a short exposure to 5-fluorouracil or a 16-hour exposure to high-specific activity H3-TdR (data not shown), an observation also reported by others.29 Thus, these results confirmed previous observations from this (data not shown) and other laboratories29 showing that most CD34+CD38low/neg cells proliferate rapidly, but asynchronously, in response to a combination of cytokines.1 The presence of MS5 cells did not change this pattern, though it might have accelerated entry into the cycles of a few cells. Coculture with MS5 cells influences the phenotype of cells produced from CD34+CD38low/neg cells that have undergone the same number of divisions A recurrent finding in these experiments was the retention by cells cultured in the presence of MS5 feeders of high levels of expression of the CD34 antigen: thus, on day 7, the proportion of CD34+ cells was 72% ± 16%, and on day 10 it was 54% ± 4% (n = 16), whereas without MS5 cells these proportions fell to 48% ± 17% on day 7 and 20% ± 2% on day 10 (P < .01). Moreover, 20% ± 2% (n = 15) of the CD34+ cells derived from cocultures on MS5 cells were still CD38low/neg (Figure 3A), whereas none of those without MS5 cells had a CD34+CD38low/neg phenotype (Figure 3B). Analysis of cultured CFSE-labeled cells on day 4 confirmed that persistence of the CD34 antigen was independent of cell division. In the absence of MS5 cells more than 50% of cells in each generation peak were CD34 , but in the presence of MS5 cells more
than 70% of cells were CD34+ (Figure 3B).
Influence of MS5 cells on the LTC-IC activity of CD34+CD38low/neg cells that have undergone the same number of divisions In the next series of experiments, we analyzed whether cells cultured for 4 days with MS5 cells and cytokines would have a different LTC-IC activity than those cultured without MS5 cells. To this end, we sorted cells that had undergone 4 or fewer divisions after 4 days in the presence or in the absence of MS5 cells, and then we cultured the sorted populations in standard LTC assays (without added cytokines)30 either in bulk cultures (2000 cells/well) or at limiting dilution (10, 50, and 200 cells/well). At least 5 independent experiments were performed for each cell generation studied.As shown in Figure 4, which summarizes
the data obtained from the bulk cultures, the number of CFCs produced
at week 5 was highly dependent on 2 parameters: the number of cell
divisions completed during the 4 days of cultures with cytokines and
the presence of MS5 cells. Independent of the culture conditions, the
number of LTC-IC-derived CFCs was the highest for cells that had
divided only twice. Thus, without MS5 cells, the geometric mean of week
5 CFCs per 1000 cells that had divided twice (2-division cells) on day
4 was 1606 (n = 5), and it decreased in cells that underwent more
divisions (to 311 for 3-division cells [n = 5] and 64 for
4-division cells [n = 9]). Corresponding numbers for MS5
cell-containing cultures were 1993, 1223, and 355, respectively. In
both conditions, with and without MS5 cells, the decrease in the number
of LTC-IC-derived CFCs between 2- division and 4-division cells was
highly significant (P < .001). Interestingly, in one experiment, cells that had divided either not at all or only once produced only 350 CFCs at week 5. Similar differences were observed in
experiments seeded with 50 cells/well (data not shown).
A striking effect of the MS5 cells was to cause a high LTC-IC activity to be retained by cells that completed 3 and 4 divisions in the first 4 days. Thus, 1223 and 355 week 5 CFCs were produced by 3-division cells with and without MS5 cells, respectively, and 311 and 64 for 4-division cells. These differences were statistically significant for both 3-division and 4-division cells (P < .01). The retention of LTC-IC activity mediated by MS5 cells was seen in each experiment, and the insert in Figure 4 shows that the mean of the increments accounted for by the presence of MS5 cells increased almost linearly with the number of cell divisions. The higher frequency of LTC-IC in 4-division cells grown on MS5 cells (1 per 50, n = 4) compared to that of cells grown without MS5 cells (1 per 300, n = 4) confirmed the above data. When CD34+CD38neg cells were used instead of
CD38low cells, the numbers of week 5 CFCs were higher. This
result was not surprising because the most immature progenitors lacked
the CD38 antigen (Table 1). Nevertheless,
in this population, the effect of MS5 cells was still clear.
A second striking observation in these experiments was the lack of a
difference in the LTC-IC activity of 2-division cells cultured with or
without MS5 cells (Figure 4). Thus 1000 cells grown without feeders
generated 1606 CFCs; with feeders they generated 1993 CFCs. One
possibility is that in these slow-dividing cells, which generated a
high number of CFC at week 5, the decline in potential induced by
cytokines was not sufficient to be translated into a decrease in the
CFC output at week 5. Consequently, an effect of MS5 cells would not be
revealed. A similar reasoning applies to cells with fewer than 2 divisions. Indeed, it has been reported that all the LTC-IC activity in
such cultures is associated with mitotically active
cells.17 If this were true, one might assume that
extending the LTC period to week 12 might unmask an effect of MS5 cells
on a more slowly recruited population. However, as shown in Table
2, this was not the case, and the CFC
output at week 12 was identical in LTC seeded with 2-division
cells initially maintained with or without MS5 cells. This result
could mean that MS5 cells have no effects on very primitive cells or
that the LTC-IC assay was not sufficiently discriminating to discern
the effects on them.
Coculture with MS5 cells retards differentiation by a direct effect on target cells Results described above indicate that MS5 cells counteract the decline in LTC-IC activity in proliferating cells that have divided 3 to 4 times in 4 days. In contrast, no effect of MS5 cells was seen in 4 days on slow-dividing cells. Additional support for this was obtained from the following experiment. Day 4 cells that had completed 2 divisions, either with or without MS5 cells, were isolated by FACS and transferred to LTC-IC assays, and the remaining cells were cultured for another 2 days with cytokines, with or without MS5 cells, as shown in Figure 1. On day 6, cells that had divided 2 more times (a total of 4 divisions from day 0) were sorted and assayed for their LTC-IC activity. Table 2 and Figure 5 summarize the results, which were reproducible in both experiments. As in previous experiments, cells that had completed 2 divisions by day 4 had similar LTC-IC activity regardless of whether MS5 cells were present (Figures 4, 5, Table 2). However, 2 days later, an effect of MS5 was clearly seen (Figure 5, Table 2). The presence of stromal cells from days 4 to 6 increased significantly the number of LTC-IC-derived CFCs compared to wells cultured without MS5 from days 4 to 6 (compare closed and open symbols in Figure 5, panels B1 and B2). Fifty percent of the wells seeded with cells cultured with MS5 from day 4 to day 6 yielded more than 100 CFCs, whereas none of those cultured without MS5 had any LTC-IC activity. Moreover, whether MS5 cells were present during the first 4 days did not affect the results on day 6 (compare Figure 5, panels B1 and B2).
Retention of LTC-IC activity depends on the number of cell divisions more than on the time in culture Finally, in a last series of experiments, we investigated whether it was the number of cell divisions completed or the time in culture that was the most important parameter causing a decline in LTC-IC activity. CD34+CD38low/neg cells were, therefore, cultured for 4 days with or without MS5 cells, and day 4 4-division cells and 0- to 3-division cells were then isolated. Four-division cells were then transferred to standard LTC (Figure 6, left panel), and 0- to 3-division cells were kept in the cytokine-containing medium (±MS5 cells) for another 2 days, after which 4-division and 0- to 3-division cells were again sorted and separately assayed for LTC-IC activity. The procedure was repeated up to day 7. In this experiment, the geometric mean of the number of week 5 CFCs was 24, 31, 40, and 10 without MS5 cells, and 44, 66, 60, and 40 with MS5 for days 4, 5, 6, and 7, respectively (Figure 6). Differences in the LTC-IC activity of cells cultured with or without MS5 cells were significant at each time point, but there was no difference in the LTC-IC activity of cells cultured for 4 to 6 days under the same conditions (ie, with or without MS5 cells). Such stability in the LTC-IC activity potential of cells that had accomplished 4 divisions either in the presence or absence of MS5 cells between day 4 and day 6 in the primary cultures indicates that, at least initially, the decline in LTC-IC activity was a function of the number of divisions completed rather that their time in culture. The decrease observed on day 7 may be partly explained by deleterious effects of repeated cell sorting and in vitro manipulation.
The alteration of stem cell fate by external signals is well established in some cellular systems,31,32 but it is still a controversial issue in the hematopoietic system,5 though recent observations strongly suggest that environmental cues might induce tissue-specific differentiation programs in stem cells.33-35 However, whether it is possible, by manipulating the environment, to change, within a given tissue, the balance between self-renewal and differentiation is less clear. Our previous studies had shown that CD34+CD38low/neg cells cultured for 10 days on murine MS5 stromal cells retained a high LTC-IC activity.25 It was unclear whether MS5 stromal cells acted by directly changing the LTC-IC activity of cells, or by altering the rate of cell division of a rare subset of cells. In this study we used fluorescent dye labeling to correlate the variation in the LTC-IC activity of CFSE-labeled CD34+CD38low/neg cells with cell division and to investigate whether stromal (MS5) cells would alter this coupling.16,18,28,36 A striking and frequent observation was the dramatic effect of MS5 cells on the LTC-IC potential of CD34+CD38low/neg marrow cells that were exposed to cytokines and that achieved 3 or 4 divisions during the 4 days of the incubation period (Figures 4, 5, Table 2). Interestingly, MS5 cells had no detectable effect on the LTC-IC activity of cells that completed only 2 divisions. Of note, the presence of MS5 at the initiation of the culture had a dramatic effect on the size of the colonies generated at week 5 by LTC-IC-derived CFCs, which were much larger, and on their ability to generate BFU-E. All these findings support the conclusion that MS5 cells counteracted the differentiation of proliferating LTC-IC. This is in agreement with our previous observation that the main effect of MS5 cells in such cultures is to increase the absolute number of LTC-IC. This may be regarded as a functional measure of their self-renewal because the LTC-IC displayed CFC outputs similar to those of the parent LTC-IC.25 These differential effects of MS5 cells on CD34+CD38low/neg cells according to their mitotic rate deserve 2 comments. First, there was a decrease in the number of week 5 CFCs with each cell generation independent of the presence of stromal cells. Thus 2-division cells on day 4 generated more than 500 and up to 2500 CFCs 5 weeks later, whereas on average 10- to 100-fold lower numbers of CFCs were produced by other cells in the same primary cultures that had divided 4 times. This loss of LTC-IC activity by cells that divide rapidly, that is more than 3 to 4 times in 4 days, was expected because cells with the highest potential are quiescent or cycle slowly.13,15 Interestingly, in one experiment, cells that did not divide at all or that divided only once within 4 days showed little LTC-IC activity (Figure 4). This suggests that LTC-IC activity may be expressed transiently by primitive cells during their differentiation and is then lost after a critical number of cell divisions.17 Accordingly, events that dictate the retention by cells of an ability to form colonies 5 weeks later may be tightly controlled, perhaps by a molecular clock, as has been described for neuronal cells37 or for hematopoietic cells in which telomere shortening has been suggested as a mechanism that determines stem cell life span.38,39 As a consequence, there may be only a narrow window in the differentiation of primitive hematopoietic cells to assess changes in LTC-IC activity. This is confirmed by data shown in Figures 4 and 5 in which the effect of MS5 cells was detectable only in cells that had gone through 3 to 4 divisions and not in more slowly dividing cells (0- to 2-division cells). Because the LTC-IC activity was very high in these 2-division cells, a subtle change induced by stromal cells may not have been translated into a change in the number of output CFCs 5 weeks later. Furthermore, in one experiment in which the culture was prolonged up to 12 weeks,40 the number of CFCs produced by cells grown with or without MS5 cells did not differ, indicating that the sensitivity of the assay was not the only parameter involved. Interestingly, the sizes and types of colonies seen in assays of LTC-IC from 2-division cells were identical, independent of the presence of MS5 cells in the primary cultures. This argues against a major functional difference in these slowly dividing LTC-ICs. Alternatively, because these cells divided only twice in 4 days, 2 mitoses might not have allowed sufficient time for MS5 cells to alter their fate. Nevertheless, it is remarkable that the increased CFC output obtained when MS5 cells were present was strongly correlated with the number of divisions completed (see Figure 4, insert). This suggests that MS5 cells act by changing the probability of a cell to retain or lose LTC-IC activity at each division. To further exclude the contribution of a rare subset of input cells induced to proliferate by MS5 cells in the observed effect, we performed a 2-step experiment. Slowly dividing (2-division) cells were sorted on day 4, and these were then incubated for 2 more days either with or without MS5 cells before assessing the LTC-IC present. The results of these experiments clearly identified a striking effect of the MS5 cells in preventing a decrease in the LTC-IC activity seen in 4-division cells exposed to cytokines alone from day 4 to day 6 (P < .01; Figure 6). Thus, it appears that any strategy to influence the LTC-IC outcome will be best identified in cells that have gone through at least 3 divisions. In summary, this study clearly shows that stromal cells can perturb the fate of primitive hematopoietic cells that actively proliferate when exposed to cytokines and that they allow some to retain primitive functions, such as those identified by the LTC-IC assay. The lack of differentiation could not be explained by a negative action of the feeder on cell proliferation.20,41 This raises the important question of the molecular mechanisms by which MS5 cells can affect the decisions of a primitive cell to lose (differentiate) or to retain (self-renew) its LTC-IC function. Increased self-renewal has been associated with increased Bcl-2 activity,42 overexpression of HoxB4,43,44 inhibition of GATA-2,45 and increased telomerase activity.46 Activation of the Notch pathway should also be considered because the Jagged-Notch interaction promotes a 2- to 3-fold expansion of normal hematopoietic progenitors by preventing the differentiation of dividing cells.12,47-49 Because MS5 cells transcribe these DSL ligands (Jagged1-2 and Delta1) (data not shown), such a mechanism is a possibility. It will also now be of interest to determine whether MS5 cells can cause the retention of properties characteristic of primitive hematopoietic cells, such as their ability to engraft NOD-SCID mice or to generate lymphoid progeny.
We thank Brigitte Izac for her expert help with the LTC-IC assays and all the surgeons and nurses who helped provide bone marrow samples. We thank the staff at Amgen-France (rhu-SCF) and Amgen-Thousands Oaks (MGDF), Immunex (Flt3-l), Novartis (IL-3), and Cilag (Epo) for their generosity in providing cytokines. We also thank Yves Levy for reading the manuscript and Pierre Walrafen for help in statistical analysis.
Submitted May 25, 2000; accepted September 11, 2000.
Supported by INSERM, grants from ARC to L.C., and by Electricité de France, Fondation de France (comité contre la leucémie). C.P. was funded by a fellowship from Fondation de France and Fédération des centres de lutte contre le Cancer.
A.B.G. and C.P. contributed equally to this work.
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: Laure Coulombel, INSERM U 474, Maternité de Port Royal, Hôpital Port Royal, 123 Boulevard de Port Royal, Paris, France; e-mail: coulombel{at}cochin.inserm.fr.
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© 2001 by The American Society of Hematology.
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K. Vanheusden, S. Van Coppernolle, M. De Smedt, J. Plum, and B. Vandekerckhove In Vitro Expanded Cells Contributing to Rapid Severe Combined Immunodeficient Repopulation Activity Are CD34+38-33+90+45RA- Stem Cells, January 1, 2007; 25(1): 107 - 114. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
1606
CFCs (n = 6) and 1993 (n = 6) CFCs, respectively, without and with
MS5 cells
MEM supplemented with 10% FCS
and 10 ng/mL PEG-recombinant human (rhu)-megakaryocyte growth and
development factor (MGDF) (a kind gift from Amgen, Thousand Oaks, CA,
and Kirin, Tokyo, Japan), 10 ng/mL rhu-Flt-3 ligand (kindly given by
Immunex, Seattle, WA), and 20 ng/mL rhu-SCF (stem cell factor) (a gift
from Amgen). Cells were cultured overnight at 37°C in 24-well plates
to allow the efflux of all CFSE not stably bound to intracellular
proteins.
