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Department of Clinical Chemistry, Microbiology, and Immunology, Ghent University Hospital, Ghent, Belgium;
Department of Hematology, Ghent University Hospital, Ghent, Belgium; and
Bloedtransfusiecentrum Oost-Vlaanderen, Ghent, Belgium
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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With this model, human hemopoietic SCID repopulating cells have been shown to reside exclusively in the CD34+38- subset, as CD34+38+ cells fail to durably engraft NOD/SCID mice (5). It was suggested that this was the result of a homing defect of CD34+38+ cells, as Kollet and colleagues (6) demonstrated that only CD34+38- and not CD34+38+ cells home to bone marrow (BM) and spleen within 1–16 h after injection, in both NOD/SCID and NOD/SCID/B2mnull mice. In contrast, other groups have shown that progenitor cells (PC) do not home by organ-specific adhesions but rather nonspecifically on the basis of organ weight and capillary bed complexity (7), suggesting that the CD34+38+ population does not contain SCID repopulating capacity due to causes other than homing, possibly proliferation defects.
Also in patients, it is not clear whether only CD34+38- cells contribute to the hemopoietic reconstitution. Fast hemopoietic recovery after transplantation of adult mobilized peripheral blood (mPB) HSC, which consists mainly of CD38+ cells (8), suggests a role for CD34+38+ cells, especially during the early posttransplant phase. These cells may be important in shortening the aplastic phase. This is further supported by clinical trials with cytokine-stimulated CD34+ cells, which show very short aplastic periods. In mice, in vivo experiments such as CFU-spleen assay show engraftment of both long-term HSC, short-term HSC, and multipotent PC. Furthermore, in vitro experiments have shown that CD34+38+ cells are multipotent and highly proliferative, but exhaust their capacity earlier (5, 9, 10, 11).
In this paper, we studied the behavior of human CD34+ cells during the early posttransplant phase and investigated whether CD34+38+ and CD34+38- cells behave differently. In the NOD/SCID mouse, we observed and analyzed different phenomena in the engraftment of CD34+ cells: 1) homing, the specific trafficking of HSC to hemopoietic organ, 2) survival or apoptosis, followed by 3) an early expansion and 4) a late expansion phase. We looked at 3 h, 24 h, 3 days, 6 days, 2 wk, and 4 wk after injection and found that after specific homing of CD34+ cells to the BM, a percentage of the cells undergo apoptosis. We further studied the short-term behavior of both CD34+38- and CD34+38+ cell populations and found that although these populations behave similarly very early after transplantation, they develop different kinetics in expansion.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mouse anti-human mAbs used were CD3 (SK7), CD34 (8G12), CD38
(HB7), and CD45 (2D1; all from BD Immunocytometry Systems, San Jose,
CA). For measuring apoptosis, annexin V (BD Immunocytometry Systems)
was used. mAbs were labeled with FITC, PE, or allophycocyanin or were
conjugated to biotin. The isotypic control Ab was IgG1 (X40; BD
Immunocytometry Systems). Anti-Fc
RII/III mAb (2.4G2, a kind gift of
Dr. J. Unkeless, Mount Sinai School of Medicine, New York, NY) was used
to block murine Fc receptors. The rat mAb against the murine IL-2R
chain was purified from supernatants of the hybridoma cell line TM-1
(kindly provided by Dr. T. Tanaka, Tokyo, Japan; Ref. 12)
grown in our laboratory (13). Before labeling, cells were
suspended in PBS containing 1% BSA and 0.1%
NaN3. For annexin V staining, cells were washed
three times with annexin V binding buffer (10 mM HEPES, 5 mM KCl, 15 mM
NaCl, 1 mM MgCl2, and 1.8 mM
CaCl2), labeled with annexin V in this buffer,
and analyzed without washing.
Mice: strain and conditioning
NOD/SCID mice were bred in our pathogen-free breeding facility
from breeding pairs originally purchased at The Jackson Laboratory (Bar
Harbor, ME). Animals were treated according to the guidelines of the
Laboratory Animal Ethical Commission of the Ghent University Hospital.
In optimization studies for human cell engraftment, the highest levels
of engraftment were obtained after pretreatment of the mice consisting
of sublethal irradiation of 350 cGy in combination with i.p. injection
of TM-1, a mAb against the murine IL-2R, to eliminate remaining
NK activity still present in NOD/SCID mice (14, 15).
However, because this pretreatment made the mice very sensitive to
GVHD, only T cell-depleted cells could be injected.
Cell sources
Umbilical cord blood (UCB) was obtained from full-term healthy newborns, and mononuclear cells were isolated within 24 h after collection by using a lymphoprep density gradient (Nycomed Pharma, Oslo, Norway) as described before (16, 17). Human adult peripheral blood stem cells were mobilized by 5 days of G-CSF treatment (Filgrastim (Amgen, Thousand Oaks, CA) or Lenigrastim (Rhone Poulenc, Lyon, France)) at a dose of 5 µg/kg q/12 h, and collected by cytapheresis. Adult mPB and adult BM were collected from healthy donors after obtaining informed consent. Child thymus (CT) was obtained from children aged 3 mo to 3 years who were undergoing cardiac surgery. All human tissue samples were obtained and used according to the guidelines of the Medical Ethical Commission of the Ghent University Hospital. Unless used fresh, cells were resuspended in 90% heat-inactivated FCS (Life Technologies, Paisley, U.K.)/10% DMSO (Serva, Heidelberg, Germany) and frozen in liquid nitrogen until use. For all experiments with CT, the cells were layered over lymphoprep gradient at 4°C after thawing to eliminate most of the dead cells. The mononuclear cell fraction from CB contained 60 ± 20% (n = 13) CD45+ cells (absolute number: 4.5 ± 2.5 x 106 CD45+ cells). The mononuclear cell fraction was stained with mouse anti-human CD3 mAb for immunomagnetic depletion by sheep anti-mouse Ig-coated beads (Dynabeads; Dynal Biotech, Oslo, Norway) with a ratio of cells:beads of 1:4. After this procedure, the percentage of CD3+ cells within the CD45+ cell fraction decreased 1 log. The average percentage of CD34+ cells after T cell depletion was 2.2 ± 1.2% of the CD45+ cells, resulting in a mean injected CD34+ cell number of 1.34 ± 0.61 x 105.
NOD-SCID repopulation assay
Mice aged 6–8 wk were given a sublethal dose of whole-body
irradiation (350 cGy, 12–15 cGy/min) with a cobalt radiation source
and injected i.p. with 200 µg of TM-1, an Ab functionally blocking
the mouse IL-2R chain.
Within 24 h after irradiation, the mice were injected i.v. with human UCB cells. Three hours, 24 h, 72 h, and 6 days after injection mice were killed and peripheral blood, thymus, lung, liver, spleen, both femora (in some experiments together with both tibia bones), and mesenteric lymph nodes were used for analysis. From these organs, cell suspensions were made and were put over a 70-µm cell strainer. RBC were lysed with hypotonic lysing buffer. Cells were counted, cell viability was checked (>85%), and after blocking the Fc receptor, cells were labeled with mAbs and analyzed on a flow cytometer.
For calculation of the total CD34+ cell number in
the BM, the number counted in both femora was multiplied by 6. If both
femur and tibia bones were used, the number counted was multiplied by
4. Two femora represent 
16% and both femora and tibiae 25% of
total murine BM (18). For liver and lung, the cell number
obtained was divided by the weight of the tissue analyzed and
multiplied by the total organ weight. For PB, the blood volume present
in the mice was estimated to be 10% of the measured body weight.
The calculated recovery of injected human CD34+ HSC is always an underestimation because of the loss of cells during injection and the loss of cells after harvesting during cell preparation (hypotonic buffer, several washing periods), which have not been taken into account.
Flow cytometry and cell sorting
Cells were analyzed or sorted on a FACSCalibur or a FACSVantage (BD Immunocytometry Systems) cell sorter, respectively, both equipped with an argon-ion laser tuned at 488 nm and a red-diode laser tuned at 635 nm. For the analysis of repopulated organs, first typically 50.000 events were acquired, of which 0.5–1% were viable propidium iodide (PI)-negative, human-CD45+ cells in the first week. Next, a live gate was set on forward scatter and fluorescence parameters (PI, CD45 FITC), and events were acquired for the remainder of the sample, to determine the percentage and absolute number of CD34+ cells. Data acquisition and analysis were done with CellQuest software (BD Immunocytometry Systems).
For sorting experiments of CD34+38+ and CD34+38-/low, UCB cells were depleted for CD3+ cells and then divided into three equal parts: one part was injected directly into mice, the second part was sorted for the CD34+38+ cell fraction and the CD34- cell fraction, and the third part was sorted for the CD34+38- cell fraction and the CD34- cell fraction. The CD34- cell fractions were both irradiated with 2500 cGy and injected together with the sorted populations to function as accessory cells.
CFSE labeling of UCB cells before injection
Cells were labeled with 0.75 µM CFSE (19) in PBS for 15 min at 37°C. After labeling, cells were washed three times in PBS supplemented with 0.1% BSA and checked on the flow cytometer for fluorescence intensity. Cells were incubated overnight in RPMI 1640 (Life Technologies) supplemented with 5% heat-inactivated FCS (Life Technologies) at 37°C (5% CO2) to enable quenching of excessive dye before injection (20).
Statistical analysis
Statistical analysis was performed with SPSS for Windows software, version 9.0. Results are expressed as the mean ± SE. Differences were evaluated with Wilcoxon signed ranks test or general linear model for repeated measures where appropriate. Statistical significance was assumed for p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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UCB CD3-depleted mononuclear cells were injected i.v., and 3
h, 24 h, 3 days, 4 days, 5 days, and 6 days after injection, the
mice were sacrificed and cell suspensions from BM, spleen, thymus,
mesenteric lymph nodes, liver, peripheral blood, and lung were analyzed
for the presence of CD34+ and as control
CD45+34- human cells.
CD45+ cells could never be detected in thymus and
mesenteric lymph nodes at this early stage. In all other organs, human
cells were detected (data not shown). Absolute numbers were calculated
as shown in Fig. 1
A. Fig. 1B shows the absolute CD34+ cell
recovery in murine organs at several time points after injection. In
liver and lung, the number of CD34+ cells was
below the detection limit. Three hours after injection, we could
retrieve 13% of the injected CD34+ cell number
in the organs studied. This number declined below 5% at day 3 (average
calculated from 19 mice injected with UCB from 7 different donors was
4.9 ± 2.9%). The majority of the CD34+
cells was found in BM, and a small amount in the spleen. Only in the BM
had the number of CD34+ cells increased
dramatically on day 6, suggesting on average a 12-fold expansion
between day 3 and day 6 for UCB-derived cells.
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C. Although the
injected population contained on average 2.2 ± 1.2%
CD34+ cells, the percentage of
CD34+ cells was enriched to 11.4 ± 6.9% in
the BM 3 h after injection, and 13.3 ± 8.9% in the spleen.
Although human nucleated cells were retrieved from liver and lung, the
percentage of CD34+ cells was lower than that of
the injected population. These relative enrichments and depletions in
CD34+ content in comparison with the injected
population at 3 h after injection suggests specific homing of the
CD34+ HSC to BM and spleen. The results obtained
at 24 h and later are more difficult to interpret because cell
death, proliferation, and redistribution may contribute to the final
outcome. Survival and expansion: CFSE labeling for cell division tracking and annexin labeling for assessment of apoptosis
The CD34+ cell number in the BM showed a dip at day 3 and started to increase again thereafter. We wanted to study this evolution more closely.
To investigate whether the increase in number of
CD34+ cells in the BM on day 6 was attributable
to proliferation or merely to delayed redistribution toward the BM of
cells initially homing in other organs, the cells were labeled with the
intracellular fluorescent dye CFSE and injected after overnight
incubation to enable quenching of excessive dye (20). As
this dye covalently couples with intracellular molecules, the dye is
divided equally between the two daughter cells on cell division, and
the number of cell divisions can be calculated from the loss in
fluorescence intensity. We observed a gradual and homogenous decrease
in the fluorescence intensity of the CD34+ cells
with time (Fig. 2A),
demonstrating that the bulk of cells underwent intense proliferation
(4–5 cell divisions by day 6).
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The evolution in the CD34+ cell number from day 1
to day 3 shows a discrepancy between the decline in
CD34+ cells and apparent proliferation of these
cells as shown by the loss of CFSE staining. To explain this, two
hypotheses were tested: redistribution from BM to other organs, or
apoptosis of a fraction of BM homed CD34+ cells.
In all murine organs, the amount of CD34+ cells
declined from day 1 to day 3. We investigated apoptosis in these cells
by using annexin V/PI staining (Fig. 2B). After 1, 2, and 3
days, on average 38, 62, and 32%, respectively, of the BM homed
CD34+ cells were annexin V positive. The
percentage of early apoptotic cells (annexin
V+/PI-) was 22, 14, and
1.5% after 1, 2, and 3 days, respectively. The remaining cells were
annexin V/PI double-positive, i.e., necrotic or late apoptotic dead
cells. By day 6, <1% of the CD34+ cells
retrieved from the BM were annexin V/PI positive. These date suggest
that after homing to the BM, CD34+ cell numbers
continue to decrease because of apoptosis and despite intense
proliferation.
Comparison of homing, survival and expansion ability of different sources of HSC
Human HSC were shown to differ in engraftment kinetics (21, 22), multilineage capacity (23, 24), and growth
characteristics (25) depending on the source of the stem
cell. Therefore, we compared the homing, survival, and expansion
characteristics of equal numbers of human CD34+
cells from UCB, CT, adult BM, and adult mPB (Fig. 3).
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, the expansion of UCB cells from
day 3 to day 6 (12-fold expansion) was significantly higher than that
of adult mPB (5.5-fold expansion) or adult BM-derived cells (4-fold
expansion), demonstrating that the proliferative capacity of UCB
CD34+ cells is superior to that of mPB or BM
CD34+ cells. One should note that although cell
numbers expanded from day 3 to day 6, the cell number recovered at day
6 was in all cases lower than the input cell number. To investigate whether CD34+ cells residing in the thymus still have the capacity to lodge in the BM and expand, we compared their homing and expansion characteristics with those of UCB, mPB, and BM CD34+ cells. Although CD34+ cells from CT did home to the BM, a gradual decrease in the amount of CD34+ cells was observed, suggesting that thymus does not contain stem cells that can survive in the BM and start expanding.
Expansion of CD34+38-/low and CD34+38+ cells
To investigate whether the described expansion from day 4 onwards
originated from CD34+38-
cells only, we sorted
CD34+38- and
CD34+38+ UCB cells (Fig. 4A) and injected them
separately into NOD/SCID mice. As a control group, we used the total
CD3-depleted cell fraction of the same UCB donor (data not shown,
similar kinetic studies with unfractionated human BM were performed by
Cashman et al.; Ref. 26). After 3 days, 1 wk, 2 wk, and 4
wk, we checked BM for the presence of human CD34+
cells. Averages ± SD from four experiments are shown in Fig. 4B. At day 3, human cells were virtually undetectable in the
BM of mice injected with
CD34+38- as well as
CD34+38+ cells (data not
shown). A week after injection of
CD34+38+ cells, these cells
could be detected in the BM and had expanded comparably to unsorted
CD3-depleted cell fractions of the same UCB source. The number of cells
in the BM kept increasing because of proliferation of both
differentiated cells and
CD34+38+ cells. However, by
4 wk after injection almost no CD34+ cells were
found in the BM of mice transplanted with
CD34+38+ population,
indicating that proliferation of
CD34+38+ cells is short
lived.
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In conclusion, we have shown that in the NOD/SCID mouse model, human CD34+ cells home specifically to the BM and spleen and that only in BM does proliferation of these cells exceeds apoptosis 3 days after injection. We also have shown for the first time engraftment of CD34+CD38+ cells and the distinct expansion kinetics properties of these cells compared with CD34+38- human UCB cells in the BM.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The initial distribution phase of CD45+ and
CD34+ cells is associated with considerable cell
loss. We measured CD34+ cell recoveries of 8
± 4% (n = 12) after 24 h and 6 ± 2%
(n = 4) after 48 h. In comparison to the data
reported in the literature, 2–3% after 24 h (7) and
<1% after 48 h (26, 27, 28), our recoveries are
relatively high. This could be explained by pretreatment of the mice
with the Ab TM-1 against the mouse IL-2R, which blocks residual
NK activity present in the NOD/SCID mouse. Tanaka et al.
(29) have investigated the in vivo effect of the TM-1
Ab injected in several mouse strains by functional and phenotyping
studies. Evaluation of NK activity in the spleen of Ab-treated mice
showed that the biological activity of the Ab lasts for at least 5 wk.
Phenotyping showed that the reduction in NK activity was the result of
the elimination of NK cells. Tournoy et al. have shown that TM-1
pretreatment of SCID mice (14) or NOD/SCID mice
(15) significantly improves survival and functionality of
human peripheral blood leukocyte grafts. Similarly, the
NOD/SCID/B2mnull mouse, which lacks NK activity,
appears to be a better recipient for human cell grafts than the
NOD/SCID mouse (30) Indeed, we observed that mice injected
with TM-1 showed a better long-term engraftment frequently
associated with thymus repopulation and
thymopoiesis.4
We demonstrated that CD34+ cells home primarily to the BM and the spleen. This is best measured at 3 h because the influence of apoptosis and proliferation is then minimized. This process has some specificity as demonstrated by the enrichment of CD34+ in total CD45+ human cell population in these organs compared with the depletion in liver and lung. This is in contrast with the data presented by Van Hennik and colleagues (7), who could not demonstrate any preferential homing of human PC, but rather tissue distribution dependent on organ size and capillary bed complexity.
Expansion rates are dependent on the origin of the stem cells: adult
BM < adult mPB < UCB. Although the slower expansion rate of
BM vs mPB did not reach significance, this observation is in agreement
with clinical data, which showed that hemopoietic recovery (defined by
days to platelet and neutrophil recovery) is faster in patients
transplanted with mPB than with BM, when optimal amounts of nucleated
cells are infused (31, 32, 33). The clinical observation that
the recovery after UCB transplantation is delayed in comparison with
mPB or even BM transplantation has been attributed to the low number of
nucleated cells and CD34+ cells infused (1 log
less than standard BM and 15 times less than standard mPB
transplantation), a homing defect, immaturity of the stem cells or the
lack of subpopulations facilitating engraftment (34). The
in vitro evidence that UCB CD34+ cells have
greater transendothelial migratory activity and increased response to
stromal-derived factor-1 and macrophage-inflammatory
protein-3 compared with mPB cells (35, 36), was
confirmed in a higher homing ability of UCB CD34+
cells in our in vivo model, although this did not reach significance.
Our data indicate that UCB cells home better and expand significantly
better than adult cells, suggesting that the low cell number or a
differentiation defect are probably important for delayed
reconstitution.
Our observations that homing is followed by a phase during which the cell number continues to drop is in contrast with previous reports from murine transplant experiments. Szilvassy et al. (37) showed in their congenic transplantation experiments that colony-forming cells show a plateau at 3 h until 24 h, but further time points were not analyzed. Lanzkron et al. (38) performed sex-mismatched syngeneic transplantation experiments and observed maximal recovery 48 h after transplantation. Different mechanisms could explain these differences. The most obvious explanation is that less apoptosis may be observed in murine than in chimeric transplantation models, as the environment for the transplanted cells may be more appropriate. Moreover, differences in mouse strain (NOD/SCID vs C57BL/6 (37, 38)) and animal conditioning (350 cGy vs 900 cGy (37) and 105 cGy (38)) could contribute to differences between our human-mouse chimeric model and the two discussed murine models.
Labeling of the cells with CFSE showed a homogenous proliferation of the CD34+ cells. Because of the small number of cells analyzed, it is possible that a minor population of nondividing cells was missed, including eventually the CD34+38- population. Although we could not exclude a minor population of nondividing cells, the bulk of the cells were already dividing shortly after injection. At all time points, CD45+CD34- cells were used as a control. In this cell fraction, both CFSE bright and CFSE dull to negative cells could be detected simultaneously, demonstrating that the loss in CFSE in the CD34+ cell fraction was not attributable to quenching of the dye. However, the cell number did not increase until after day 3. This could be explained by either redistribution of the CD34+ cells or apoptosis of a fraction of the BM-homed cells. Our experiments did not provide arguments for organ redistribution, as in all murine organs examined, the number of CD34+ cells declined between day 1 and day 3. Staining of the BM-homed cells at these time points with annexin V and PI shows a large fraction of the cells in apoptosis at days 1, 2, and 3. The percentage of early apoptotic cells within these annexin-positive cell fractions decreased day by day, in that at day 3 >90% of all annexin-positive cells also were PI positive. By day 6, <1% of the CD34+ cells retrieved from the BM were annexin positive. To investigate whether CFSE could leak out of apoptotic cells, we induced apoptosis by irradiation (3000 cGy) in UCB mononuclear cells that were labeled with CFSE 15 h before. We found that apoptotic cells maintain CFSE high (data not shown). These observations indicate that only a very small fraction of the cells present at 3 h is responsible for early expansion and engraftment. This is in agreement with reports in both mice and human that long-term reconstitution is oligoclonal (39, 40).
In this paper, we have shown that both
CD34+38+ and
CD34+38- cells proliferate
on injection. However, human hemopoietic SCID repopulating cells have
been shown to reside exclusively in the
CD34+38- subset, as
CD34+38+ cells fail to
durably engraft NOD/SCID mice (5, 41). Little is known
about the short-term engraftment characteristics of
CD34+ subsets in the NOD/SCID mouse. Kollet et
al. (6) demonstrated that exclusively
CD34+CD38-/low and not
CD34+CD38+/high home to BM
and spleen within 1–16 h after injection in both NOD/SCID and
NOD/SCID/B2mnull mice. Even 16–24 h after
transplantation,
CD34+CD38+/high cells could
not be detected in murine BM and spleen. In contrast, our data show
that both cell populations home to BM and spleen. It is possible that
the discrepancy is technical in nature: a difference in CD38 Ab
intensity or gating strategy for CD38-/low vs
CD38+/high. Moreover, injection of the mice with
TM-1 reduces xenoreactivity by NK cells and may thereby allow
CD34+38+ cells to
home.
Both short- and long-term data cited above (5, 6, 41) indicate that only CD34+38- cells are important for engraftment also immediately after transplantation. However, this seems to contradict both in vitro and clinical data. In vitro data have shown that the CD34+CD38+ fraction contains colony-forming cells and long-term culture-initiating cells and that these develop more quickly but are more quickly exhausted than from CD34+38- cells (5, 9, 10). Transplantation of mPB HSC, which mainly consist of CD34+CD38+ cells (8), results in fast reconstitution, and it is reasonable to assume that these early developing cells are the progeny of the bulk of the cells. Moreover, in vitro expansion with both early- and late-acting growth factors of purified HSC resulted in significant shortening of the aplastic phase. Such short aplastic phases could not be achieved by elevation of the HSC dose only (42, 43, 44). We showed here that this CD34+38+ cell population does home and starts to proliferate very quickly, and therefore may contribute to the early reconstitution after stem cell transplantation. This expansion of CD34+38+ cells gradually fades out, and CD34+38+ cells (but not their progeny) become nearly undetectable 4 wk after injection, which is in line with the inability of CD34+38+ cells to sustain reconstitution of NOD/SCID mice (5, 41).
Our data are in line with the data of Zijlmans et al. (45), who distinguished three separate subpopulations of murine PCs, i.e., committed PCs, which have no role in radioprotection, stem cells with short-term repopulating ability, and stem cells with long-term repopulating ability.
Recently, CD34-Lin- cells were identified as a promising repopulating stem cell population (46). In addition, it has been reported that CD34+ cells can down-modulate CD34 (47). It would be interesting to address these issues by studying the short-term behavior of CD34- cells in our model. However, the small number of cells and the need for some additional membrane markers to distinguish between murine cells and human CD34-Lin- cells did not allow us to address this issue in the present study.
In this paper, we have experimentally defined homing, survival, and expansion of human CD34+ cells and have shown that homing does not necessarily lead to survival and subsequent expansion (CT CD34+ cells) and that early expansion does not always result in long-term engraftment (CD34+38+ cells). At the moment, studies on the influence of culture conditions, blocking Abs, and phenotype on engraftment of HSC consider long-term engraftment in the NOD/SCID mouse (12–15 wk) as the only end point. Here we have shown that to study all phases of engraftment, four time points have to be studied: 3 h for homing, 3 days for the net result of early proliferation and apoptosis, 1 wk for the expansion of the CD34+38+ cells, and 3–4 wk for expansion of the CD34+38- cells.
In conclusion, we have developed a model that makes it possible to study the short-term behavior of transplanted human HSC. We have shown that CD34+ cells home specifically to BM and spleen and only lodge and start to expand in the BM, with kinetics depending on the expression of CD38. This model not only significantly shortens the engraftment model for studying CD34+38- cells but also makes it possible to study the homing, survival, and expansion of CD34+38+ cells that are important for the short-term engraftment in murine models and which may be important in shortening of the aplastic phase in the clinical setting.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Tessa C. C. Kerre, Department of Clinical Chemistry, Microbiology and Immunology, Ghent University Hospital, 4BlokA, De Pintelaan 185, B-9000 Gent, Belgium. E-mail address: Tessa.Kerre{at}rug.ac.be
3 Abbreviations used in this paper: HSC, hemopoietic stem cells; BM, bone marrow; PC, progenitor cells; mPB, mobilized peripheral blood; UCB, umbilical cord blood; CT, child thymus; PI, propidium iodide.
4 T. Kerre, G. De Smet, M. De Smedt, F. Offner, B. Vandekerckhove, and J. Plum. An adapted NOD/SCID model supports the development of phenotypically and functionally mature T cells from umbilical cord blood CD34+ cells. Submitted for publication.
Received for publication April 16, 2001. Accepted for publication July 23, 2001.
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References |
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-chain. Characterization of receptor expression in normal lymphoid cells and EL-4 cells. J. Immunol. 147:2222.[Abstract]
3 thymocytes: mature but not immature V3 cells require endogenous IL-2 and IL-7 to survive: evidence for cytokine redundancy. Int. Immunol. 7:843.-chain blockade improves human leukocyte engraftment in SCID mice. Eur. J. Immunol. 28:3221.[Medline]
-chain monoclonal antibody. J. Exp. Med. 178:1103. compared with mobilized adult progenitor cells. Br. J. Haematol. 107:441.[Medline]
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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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T. Lapidot, A. Dar, and O. Kollet How do stem cells find their way home? Blood, September 15, 2005; 106(6): 1901 - 1910. [Abstract] [Full Text] [PDF]
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J. L. McKenzie, O. I. Gan, M. Doedens, and J. E. Dick Human short-term repopulating stem cells are efficiently detected following intrafemoral transplantation into NOD/SCID recipients depleted of CD122+ cells Blood, August 15, 2005; 106(4): 1259 - 1261. [Abstract] [Full Text] [PDF]
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H. Glimm, M. Schmidt, M. Fischer, K. Schwarzwaelder, M. Wissler, S. Klingenberg, C. Prinz, C. F. Waller, W. Lange, C. J. Eaves, et al. Efficient marking of human cells with rapid but transient repopulating activity in autografted recipients Blood, August 1, 2005; 106(3): 893 - 898. [Abstract] [Full Text] [PDF]
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T. Byk, J. Kahn, O. Kollet, I. Petit, S. Samira, S. Shivtiel, H. Ben-Hur, A. Peled, W. Piacibello, and T. Lapidot Cycling G1 CD34+/CD38+ Cells Potentiate the Motility and Engraftment of Quiescent G0 CD34+/CD38-/low Severe Combined Immunodeficiency Repopulating Cells Stem Cells, April 1, 2005; 23(4): 561 - 574. [Abstract] [Full Text] [PDF]
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M. Xu, E. Bruno, J. Chao, H. Ni, V. Lindgren, R. Nunez, N. Mahmud, G. Finazzi, S. M. Fruchtman, U. Popat, et al. The constitutive mobilization of bone marrow-repopulating cells into the peripheral blood in idiopathic myelofibrosis Blood, February 15, 2005; 105(4): 1699 - 1705. [Abstract] [Full Text] [PDF]
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 H. E. Daldrup-Link, M. Rudelius, G. Piontek, S. Metz, R. Brauer, G. Debus, C. Corot, J. Schlegel, T. M. Link, C. Peschel, et al. Migration of Iron Oxide-labeled Human Hematopoietic Progenitor Cells in a Mouse Model: In Vivo Monitoring with 1.5-T MR Imaging Equipment Radiology, January 1, 2005; 234(1): 197 - 205. [Abstract] [Full Text] [PDF]
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R. Gimeno, K. Weijer, A. Voordouw, C. H. Uittenbogaart, N. Legrand, N. L. Alves, E. Wijnands, B. Blom, and H. Spits Monitoring the effect of gene silencing by RNA interference in human CD34+ cells injected into newborn RAG2-/- {gamma}c-/- mice: functional inactivation of p53 in developing T cells Blood, December 15, 2004; 104(13): 3886 - 3893. [Abstract] [Full Text] [PDF]
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F. Ahmed, S. J. Ings, A. R. Pizzey, M. P. Blundell, A. J. Thrasher, H. T. Ye, A. Fahey, D. C. Linch, and K. L. Yong Impaired bone marrow homing of cytokine-activated CD34+ cells in the NOD/SCID model Blood, March 15, 2004; 103(6): 2079 - 2087. [Abstract] [Full Text] [PDF]
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F. Mazurier, O. I. Gan, J. L. McKenzie, M. Doedens, and J. E. Dick Lentivector-mediated clonal tracking reveals intrinsic heterogeneity in the human hematopoietic stem cell compartment and culture-induced stem cell impairment Blood, January 15, 2004; 103(2): 545 - 552. [Abstract] [Full Text] [PDF]
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X. Wang, M. Rosol, S. Ge, D. Peterson, G. McNamara, H. Pollack, D. B. Kohn, M. D. Nelson, and G. M. Crooks Dynamic tracking of human hematopoietic stem cell engraftment using in vivo bioluminescence imaging Blood, November 15, 2003; 102(10): 3478 - 3482. [Abstract] [Full Text] [PDF]
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P. A. Plett, S. M. Frankovitz, and C. M. Orschell Distribution of marrow repopulating cells between bone marrow and spleen early after transplantation Blood, September 15, 2003; 102(6): 2285 - 2291. [Abstract] [Full Text] [PDF]
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 J. Cashman, B. Dykstra, I. Clark-Lewis, A. Eaves, and C. Eaves Changes in the Proliferative Activity of Human Hematopoietic Stem Cells in NOD/SCID Mice and Enhancement of Their Transplantability after In Vivo Treatment with Cell Cycle Inhibitors J. Exp. Med., November 4, 2002; 196(9): 1141 - 1150. [Abstract] [Full Text] [PDF]
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M. Schmidt, P. Zickler, G. Hoffmann, S. Haas, M. Wissler, A. Muessig, J. F. Tisdale, K. Kuramoto, R. G. Andrews, T. Wu, et al. Polyclonal long-term repopulating stem cell clones in a primate model Blood, September 26, 2002; 100(8): 2737 - 2743. [Abstract] [Full Text] [PDF]
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 M. De Smedt, K. Reynvoet, T. Kerre, T. Taghon, B. Verhasselt, B. Vandekerckhove, G. Leclercq, and J. Plum Active Form of Notch Imposes T Cell Fate in Human Progenitor Cells J. Immunol., September 15, 2002; 169(6): 3021 - 3029. [Abstract] [Full Text] [PDF]
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T. C. C. Kerre, G. De Smet, M. De Smedt, A. Zippelius, M. J. Pittet, A. W. Langerak, J. De Bosscher, F. Offner, B. Vandekerckhove, and J. Plum Adapted NOD/SCID model supports development of phenotypically and functionally mature T cells from human umbilical cord blood CD34+ cells Blood, March 1, 2002; 99(5): 1620 - 1626. [Abstract] [Full Text] [PDF]
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,
dashed line) and both early and late apoptotic/necrotic fraction
(annexin+, 
, full line) within the BM-homed
CD34+ cells. Data shown are mean values SDs from four
experiments with four different UCB donors. In each experiment, at
least two mice were examined at each time point.
