|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
HEMATOPOIESIS
From the Departments of Medicine and Pediatrics, Herman
B Wells Center for Pediatric Research and the Department of
Microbiology/Immunology, Indiana University School of Medicine,
Indianapolis.
Differences in engraftment potential of hematopoietic stem cells
(HSCs) in distinct phases of cell cycle may result from the inability
of cycling cells to home to the bone marrow (BM) and may be influenced
by the rate of entry of BM-homed HSCs into cell cycle. Alternatively,
preferential apoptosis of cycling cells may contribute to their low
engraftment potential. This study examined homing, cell cycle
progression, and survival of human hematopoietic cells transplanted
into nonobese diabetic severe combined immunodeficient (NOD/SCID)
recipients. At 40 hours after transplantation (AT), only 1% of
CD34+ cells, or their G0
(G0CD34+) or G1
(G1CD34+) subfractions, was detected in the BM
of recipient mice, suggesting that homing of engrafting cells to the BM
was not specific. BM of NOD/SCID mice receiving grafts containing
approximately 50% CD34+ cells harbored similar numbers of
CD34+ and CD34 Homing of transplanted hematopoietic stem cells
(HSCs) to the bone marrow (BM) of recipient animals is an essential
step in engraftment and maintenance of hematopoiesis. It is believed
that homing, a process defined as migration of cells to target tissues, directs cells during ontogeny from the yolk sac and/or para-aortic splanchnopleura to the fetal liver and finally to fetal
BM1-4 where hematopoiesis is maintained throughout adult
life. However, processes governing trafficking, migration, and homing
of HSC, both at the molecular and cellular levels, remain poorly
understood. Although some studies lend support to the involvement of
particular adhesion molecules in both homing and egress of HSCs to and
from the BM,5-10 other studies challenge the concept of
directed homing to the BM and maintain that homing is a nonspecific
process.11,12 Under such circumstances, it is believed
that transplanted HSCs are distributed in the host on the basis of
organ mass and complexity of the capillary bed of each
organ.11 Nonetheless, directed trafficking to the BM of
certain phenotypes of hematopoietic progenitor cells (HPCs), but not
others, has been documented.13 Similarly, enhanced
engraftment potential of candidate HSCs expressing a particular
repertoire of adhesion molecules has been
demonstrated.14,15
On trafficking to the marrow, HSCs lodge specifically in and become
anchored to specialized niches of the marrow microenvironment where
they undergo massive cell proliferation, resulting in the establishment
and maintenance of a new hematopoietic system. Murine studies examining
the fate of transplanted cells in nonablated hosts documented that
as early as 12 hours after transplantation (AT) in excess of 50% of
long-term engrafting cells enter active phases of cell
cycle.16 Together, homing studies and those examining the
fate of transplanted HSCs suggest that cells incapable of supporting
long-term engraftment may suffer from 2 distinct, but not mutually
exclusive, defects, precluding them from either homing to the BM or
entering active phases of cell cycle. The latter potential defect is
rather intriguing in light of our previous demonstration that
engrafting mobilized peripheral blood cells (those capable of
sustaining hematopoiesis in an appropriate host) are predominantly
found in the G0 phase of cell cycle, whereas those in
G1 are almost devoid of marrow repopulating
potential.17 This finding suggests that following
transplantation, cells in G0 begin to divide after homing
to the BM, whereas proliferation of BM-homed G1 cells is
halted. Alternatively, it is perceivable that, when examined head to
head, engrafting cells home to the BM where they can be detected, while
nonengrafting cells fail to traffic to the marrow and initiate hematopoiesis.
Although these hypotheses may be theoretically easy to examine in a
transplantation model, realistically, technical difficulties involved
in tracking and isolating small numbers of candidate HSCs in recipient
animals contributed to the near complete absence of such studies.
However, recent advances in the use of xenogeneic transplantation
models11,18 or tracking of transplanted cells with
fluorescent dyes19-21 made such investigations possible.
Only one recent study21 examined proliferation kinetics of
transplanted murine cells shortly after transplantation and assessed
comparatively the homing efficiency of engrafting versus nonengrafting phenotypes.
In the absence of direct and conclusive measurements of these
parameters for human HSCs, we designed a series of experiments to
evaluate the homing efficiency, cell cycle kinetics, and survival of
quiescent and cycling human CD34+ cells transplanted into
conditioned nonobese diabetic severe combined immunodeficient
(NOD/SCID) recipients. To better understand the dynamics of HSC homing
and engraftment under different transplantation conditions, the fate of
freshly isolated and ex vivo-expanded BM and mobilized peripheral
blood (MPB) CD34+ cells or fractions of
CD34+ cells was examined. We report that homing of fresh or
ex vivo-expanded cells to the BM microenvironment is not specific and
that immediately after transplantation BM-homed cells remain quiescent
for a considerable period of time regardless of the phenotype or cell
cycle status of these cells.
Collection, purification, and fractionation of human
CD34+ cells
Cell staining and sorting
Short-term culture and staining with CFSE Selected (not purified) CD34+ cells were cultured in complete medium consisting of Iscoves modified Dulbecco medium (BioWhittaker) supplemented with 10% FCS, 2 mM L-glutamine (BioWhittaker), and antibiotics (penicillin 100 U/mL and streptomycin 100 µg/mL; BioWhittaker). Cells were cultured at 0.5 × 106 cells/mL and were supplemented with a 6-cytokine combination containing human stem cell factor (SCF) at 100 ng/mL, Flt-3 ligand at 50 ng/mL, megakaryocyte growth and development factor (MGDF) at 50 ng/mL, interleukin-3 (IL-3) at 100 ng/mL, IL-6 at 100 ng/mL, and granulocyte-macrophage colony-stimulating factor (GM-CSF) at 20 ng/mL. All cytokines were a kind gift from Amgen (Thousand Oaks, CA). Cytokines were added at the initiation of the culture and every 48 hours thereafter. Between days 5 and 7, cells were harvested, washed, and counted. A sample was immunophenotyped to determine the percentage of CD34+ cells. When needed, cells were stained with Hst only as described above and sorted to yield cells in G0/G1 or in S/G2+M as previously described.15Fresh or cultured cells were stained with the protein dye CFSE before transplantation. Cells were washed and resuspended at less than 5 × 106 cells/mL in serum-free HBSS. CFSE (Molecular Probes) was added at 1 µM, and the cells were incubated for 10 minutes at 37°C. Uptake of the dye was stopped by the addition of chilled HBSS containing 10% FCS, and the cells were washed in this medium 3 times. Cells were resuspended in complete medium and, when possible, were incubated 2 to 4 hours at 37°C to allow excess dye to efflux. Cells were then washed and resuspended for transplantation. Transplantation of test cells into NOD/SCID mice NOD/LtSz-scid/scid (NOD/SCID) mice23 were bred and housed at Indiana University and were kindly provided by Dr D. A. Williams (Indianapolis, IN). Mice were housed in microisolators under pathogen-free conditions and received autoclaved food and acidified water ad libitum. Animal experiments were performed in accordance with institutional guidelines approved by the Animal Care Committee of Indiana University School of Medicine. Nine- to 12-week-old NOD/SCID mice were sublethally irradiated with 3 Gy from a 137Cs source. Mice received by intravenous injection as accessory cells between 8 and 12 × 106 nonadherent CD34
adult BM or MPB cells irradiated with 80 Gy and were transplanted 2 to
24 hours later. Between 6 × 105 and
3 × 106 cells were transplanted per recipient, depending
on the availability of cells. All mice in the same experiment received
grafts containing equal numbers of cells. At specific time points AT
(up to 72 hours AT), blood was collected from the retro-orbital plexus.
Mice were killed by cervical dislocation, and the spleen and BM were
collected. Single-cell suspensions were prepared from these tissues,
and red blood cells were lysed in 0.155 M NH4Cl, 0.01 M
KHC03, and 0.1 mM EDTA. Cell suspensions were washed twice
and resuspended in Iscoves modified Dulbecco medium with 10% FCS for
further analysis. Because of the effect of radiation, cellularity of
recipient BM and spleen declined 48 hours AT to 10% to 25% of that
observed in nonirradiated mice.
Analysis of homing and isolation of human chimeric cells Human cells in the BM, spleen, or blood of recipient mice were detected flow cytometrically by visualization of CFSE-stained cells over a background of unlabeled murine cells (Figure 1). The bright fluorescence of CFSE was sufficient to separate labeled human cells from unlabeled murine cells by at least 1 log. When required, murine BM or spleen cells were stained with PE- or APC-conjugated antihuman CD34 monoclonal antibodies to permit the isolation of CFSE+CD34+ and CFSE+CD34 cells by cell sorting (Figure 1).
Percentages of CFSE+ cells were used to calculate total
numbers of human cells recovered by using cell counts from the spleen
and adjusted cell counts from the BM, depending on the bones harvested
for analysis.24 Percentage of CFSE+ cells was
calculated from listmode files containing between 105 and
5 × 105 events after light scatter gating of small and
large cells and exclusion of debris and red cells. For cell sorting,
all the cells from an individual tissue were sorted when needed, or
sorting was halted when enough donor-derived cells were collected for further analysis.
Cell cycle analysis Cell cycle status of test cells was determined by the analysis of propidium iodide (PI)-stained cells as previously described.25 Cell pellets were stained for 30 minutes with equal volumes of phosphate-buffered saline (PBS) containing 0.1 mg/mL PI (Behring Diagnostics, La Jolla, CA) and 0.6% Nonidet P-40 and 2 mg/mL RNAse (both from Sigma) prepared in PBS. Cell cycle analysis was performed on a FACScan (BDIS), and data were analyzed by using CellQuest software (BDIS).Assessment of apoptosis Various cell fractions (CFSE+ cells isolated from recipients or cells cultured with or without cytokine supplementation) were evaluated for apoptosis by the analysis of Annexin V-positive cells. Cell pellets were stained with APC-conjugated CD34 when needed and PE-conjugated Annexin V (BDIS) at 4°C for 20 minutes. Cells were washed once in DMEM (BioWhittaker) supplemented with 15% FCS. Cells were resuspended in 200 µL DMEM and 10 µL PBS containing 10 µg/mL PI for assessment of dead cells. Analysis was performed on a FACSCalibur (BDIS), and data were analyzed by using Cellquest software. Only CFSE+PI cells were considered for
analysis of CD34 and Annexin V.
Statistical analysis Data are reported as mean ± SD and were analyzed by using a Student t test. Differences yielding P values < .01 were considered statistically significant.
Preliminary studies indicated that the highest degree of cell recovery from the BM and spleen of transplantation recipients was between 36 and 48 hours AT (data not shown). This observation is consistent with that of Lanzkron et al21 who determined that percentage of PKH26-labeled murine cells from syngeneic recipients was maximal at 48 hours AT. As such, all subsequent analyses (unless indicated otherwise) were performed between 40 and 48 hours AT and are reported as 40 hours. A very small number of CFSE+ cells was detected in the peripheral blood of recipient animals 24 hours AT (< 0.05%), and at 40 hours AT transplanted cells were almost undetectable in the blood. Therefore, data from the analysis of peripheral blood will not be presented. A total of 34 experiments using 1 to 3 animals per group were performed. Data from like experiments were pooled. Cell cycle status of BM- and spleen-homed CD34+ cells As expected, freshly isolated MPB and BM CD34+ cells were predominantly in G0/G1 phases of cell cycle (99.2% ± 0.6% and 88.4% ± 3.7%, respectively; Figure 2). When maintained in short-term culture for up to 48 hours, these cells exited G0/G1 efficiently, and less than 66% of either population was in G0/G1. However, cells recovered from NOD/SCID mice 40 hours AT were quiescent with more than 82% of either population in the BM or spleen of these recipients detected in G0/G1 (Figure 2).
Effect of in vivo administration of cytokines on cell cycle kinetics of transplanted cells Results shown in Figure 2 provoked the question of whether transplanted human cells receive adequate cytokine stimulation in the mouse microenvironment to induce proliferation shortly after transplantation. To investigate this question, recipient mice were injected intraperitoneally with a cocktail of 7 human cytokines (see legend of Figure 3 for details) on days 2, 1, 0, and +1 relative to the transplantation of test cells that
was performed, according to this schema, on day 0. Mice were killed, and BM and spleen cells were collected for analysis 24 and 48 hours AT.
As can be seen in Figure 3 regardless of the tissue analyzed,
transplanted cells remained quiescent in cytokine-treated recipients to
the same level observed in control mice even at 48 hours AT. It is
important to point out that cells maintained in vitro for 48 hours with
a similar cytokine cocktail mixture (with adjusted concentrations for
in vitro culture) were capable of exiting G0/G1
and progressing normally through active phases of cell cycle
(Figure 3).
Cell cycle status of BM- and spleen-homed ex vivo-expanded CD34+ cells That transplanted CD34+ cells, even when stimulated with exogenously administered cytokines, do not enter into active phases of cell cycle in recipient mice prompted us to examine whether transplanted cycling cells continue their cell cycle progression after lodgment in the BM. To that effect, MPB and BM CD34+ cells were first cultured in vitro for 5 to 6 days in the presence of 6 cytokines. These expanded cells were harvested, stained with CFSE, and transplanted into conditioned NOD/SCID mice. As can be seen in Figure 4, expanded MPB and BM CD34+ cells were actively cycling by day 5 with 65% and 70% in G0/G1, respectively. In contrast, in excess of 91% of either MPB- or BM-expanded CD34+ cells isolated from either the BM or spleen of recipient mice 40 hours AT were in G0/G1 (Figure 4), suggesting that cycling of these cells was arrested after their lodgment in these organs. Cells maintained in culture continued to cycle normally (Figure 4). Whether in vivo administration of exogenous cytokines prevents cell cycle arrest of transplanted ex vivo-expanded cells was not investigated.
Cell cycle status of BM- and spleen-homed ex vivo-expanded CD34+ cells in different phases of cell cycle Because transplanted expanded cells contained cells in different phases of cell cycle, it was not possible to determine whether cycling cells trafficked to the BM and to what degree the cell cycle status of BM-homed cells reflected that of cycling or quiescent cells initially contained in the graft. To circumvent these limitations, expanded cells were stained with Hst on days 5 to 7 and sorted to yield cells in G0/G1 or in S/G2+M that were then transplanted and assayed 40 hours later. Data generated from these studies are shown in Figure 5. Cultured cells isolated in G0/G1 or S/G2+M (purities = 85.0% and 76.0%, respectively; Figure 5) progressed through cell cycle as expected when maintained in vitro. Only 69% of sorted G0/G1 cells remained in G0/G1 at 40 hours later (P < .01, between sorted G0/G1 cells and same cells in culture 40 hours later). Cells in S/G2+M continued their cycle such that at 40 hours 59% of these cells were in G0/G1 (P < .01, between sorted S/G2+M cells and same cells in culture 40 hours later). However, between 84.7% ± 11.3% and 95.1% ± 3.2% of cells recovered from the BM and spleen of recipient mice 40 hours AT were in G0/G1 (Figure 5), including those which 40 hours earlier were predominantly in S/G2+M phases of cell cycle (P < .01, between sorted S/G2+M cells and cells recovered from BM and spleen 40 hours AT). Of interest is that there was no difference in the percentage of cells in G0/G1 among sorted G0/G1 cells before transplantation (84.7%) and AT (88.7% and 95.1% in BM and spleen, respectively).
To follow more closely cell cycle progression of transplanted
cells in G0/G1 or S/G2+M,
experiments designed to assay cell cycle status of transplanted cells
at 3, 16, and 40 hours AT were conducted (Table
1). In a design similar to that described
above, expanded BM cells in G0/G1 or
S/G2+M were transplanted and recovered from the BM of
NOD/SCID mice at 3 time points. As expected,
G0/G1 cells remained quiescent in the BM of
recipient mice throughout the observation period while in culture these
cells progressed into S/G2+M normally. However, in the
absence of exogenous cytokines in vitro, these cells remained dormant
(Table 1). Surprisingly, although a fair number of S/G2+M
cells maintained in culture with cytokines progressed into
G0/G1 16 hours later and most likely traversed
again into S/G2+M by 40 hours (63.7% in
G0/G1), similar cells in vivo were unable to
reenter S/G2+M and were found predominantly in
G0/G1 at 40 hours AT. Of interest is that
S/G2+M cells maintained in vitro without cytokines
progressed normally into G0/G1 (Table 1),
confirming that cells that had progressed beyond the G1
checkpoint are capable of going into mitosis in the absence of
mitogenic signals.
Cell cycle status of BM- and spleen-homed G0CD34+ and G1CD34+ cells Investigations into cell cycle progression of transplanted cells residing in either G0/G1 or S/G2+M did not establish whether cell cycle arrest of these cells was a specific phenomenon restricted to the behavior of expanded cells. Thus, these studies left unanswered the question of whether freshly isolated CD34+ cells in different phases of cell cycle behaved similarly in vivo. G0CD34+ and G1CD34+ cells from MPB were isolated as previously described17 and transplanted into conditioned NOD/SCID recipients. Because of the lack of substantial numbers of cells in S/G2+M in MPB26-28 and to the limited number of these cells that can be isolated from fresh BM CD34+ cells, these analyses were restricted to MPB G0 and G1CD34+ cells (Figure 6). Not only do G0 and G1 fractions of MPB CD34+ cells represent 2 mitotically distinct groups of cells, they also segregate CD34+ cells into NOD/SCID mice engrafting (G0CD34+ cells) and nonengrafting (G1CD34+ cells).17 In addition, such a separation provides a sizable enrichment for CD34+CD38 cells within
G0CD34+ cells.29 To follow cell
cycle progression more closely, cells were analyzed at 24, 48, and 72 hours AT, when possible. As can be seen in Figure 6, both
G0 and G1CD34+ cells began to exit
G0/G1 at 24 hours (88.5% and 83.7% in
G0/G1, respectively) and cycled effectively
after 48 hours of in vitro culture such that between 53.4% and 61.3%
of these cells were in G0/G1 between 48 and 72 hours. However, most G0CD34+ cells recovered
from the BM of recipient mice persisted in
G0/G1 especially 24 hours AT (96.4%), and, by
48 hours, 87.8% remained in G0/G1.
Interestingly, a higher percentage of G0CD34+
cells trafficking to the spleen remained in
G0/G1 at all time points examined (Figure 6).
The difference between the percentage of transplanted G0
cells remaining in G0/G1 48 hours AT (87.8%) and that among similar cells maintained in culture (56.0%) was statistically significant (P < .01). Although a smaller
fraction of G1CD34+ cells homing to the BM
remained in G0/G1 48 hours AT (78.1%), the
difference in percentage of quiescent cells contained in this group and
the corresponding fraction among cells maintained in culture was
significant (P < .01).
Trafficking of CD34+ cells to the BM of recipient mice Transplantation of grafts composed of enriched, but not purified, CD34+ cells allowed for the determination of the percentage of CD34+ cells, among total transplanted cells, trafficking to the BM and spleen of recipient mice. This percentage was compared with that detected in the graft to assess whether CD34+ cells preferentially homed to the BM or spleen of recipient mice (Figure 7). On average, 55.4% ± 13.3% of transplanted fresh BM cells were CD34+. Cells collected from the BM of recipient mice contained 56.9% ± 27.0% CD34+, whereas those trafficking to the spleen were composed of 34.3% ± 15.4% CD34+ cells (Figure 7). These results suggest that trafficking of CD34+ cells to the BM is not specific, but that a substantially larger percentage of CD34+ cells home to the BM compared with the spleen. To investigate whether homing of ex vivo-expanded CD34+ cells is compromised because of biologic and/or physiologic changes of CD34+ cells in vitro, similar analyses were performed on transplanted ex vivo-expanded cells. Grafts of expanded CD34+ cells contained 25.2% ± 8.6% CD34+ cells. BM-homed cells contained 24.3% ± 5.7% CD34+ cells, whereas 11.8% ± 6.3% CD34+ cells were detected in spleen-homed cells (Figure 7). These data illustrate that homing of expanded CD34+ cells, measured as the relative percentage of cells detected in the BM at a given time point AT, is not compromised compared with fresh cells.
Trafficking of CD34+ cells AT was also analyzed by
calculating the number of cells recovered in the BM and spleen of
recipient mice as a percentage of the original number contained in the
graft. Data shown in Figure 8 illustrate
that between 1.0% and 1.1% of transplanted total, or
CD34+, or CD34
Fate of BM-homed graft cells in different phases of cell cycle Having observed that cycling as well as quiescent cells were predominantly in G0/G1 phases of cell cycle 40 hours AT, we hypothesized that cycling cells might be more adversely affected by an unscheduled cell cycle arrest than quiescent cells. Under these circumstances, cells in G0 and early G1 may remain quiescent in the BM microenvironment until appropriate signals and conditions are conducive for their proliferation, whereas interruption of cell cycle progression of cycling cells may lead to apoptosis. We examined this hypothesis by transplanting into conditioned NOD/SCID recipients CFSE-stained ex vivo-expanded MPB or BM CD34+ cells separated, before transplantation, into cells in G0/G1 or in S/G2+M. BM-homed cells were recovered 40 hours AT, and the percentage of Annexin V-positive cells was determined. Results from 3 separate experiments are shown in Figure 9. While only 17.1% ± 6.3% of cells recovered from recipients of xenografts containing G0/G1 cells displayed signs of programmed cell death, more than one third of transplanted S/G2+M cells (34.6% ± 5.9%, P < .01) were apoptotic, demonstrating that cell cycle arrest of these cells in the BM of recipient mice was detrimental to their long-term survival. Of interest is that only 15.1% ± 6.3% of S/G2+M cells maintained in complete medium for 40 hours without cytokine supplementation were apoptotic, a percentage similar to that observed for cells in G0/G1 when treated similarly (17.2% ± 7.2%, P = .7). When supplemented with cytokines, less than 4% of both phenotypes of cells were apoptotic 40 hours later.
Engraftment and initiation of hematopoiesis by transplanted HSCs depend on 3 important criteria. First, transplanted cells must home to the BM microenvironment. Second, HSCs must lodge in the appropriate niches of the microenvironment, and, third, these cells must possess both the proliferative and multilineage differentiation potentials required for establishment of hematopoiesis. Many aspects of all 3 phases of engraftment of human stem and progenitor cells are poorly explored and ill defined. To shed some light on the first and third phases of this paradigm of hematopoietic engraftment, we examined homing patterns of transplanted human hematopoietic cells in a xenogeneic transplantation model and investigated cell cycle status of these cells shortly after their lodgment in the BM microenvironment. Our observations suggest that homing of transplanted hematopoietic cells to the BM is not specific but that subsequent lodgment in the BM microenvironment of engrafting cells may be selective. Furthermore, we documented that HPCs retained in the BM remain quiescent for the first 40 hours AT possibly due to an active proliferation inhibition mechanism that is yet to be investigated. Determination of whether homing of engrafting cells to the BM
microenvironment is specific would have been best examined by assessing
the marrow repopulating potential of BM-homed cells in secondary
recipients. However, the scarcity of cells recovered from the BM of
primary recipients relative to what may be required to sustain
measurable engraftment precluded such investigations. Nevertheless, our
conclusion regarding nonspecificity of homing was inferred from the
collective behavior of different fractions of CD34+ and
CD34 Whereas in mouse-to-mouse transplantation studies,11,19-21,32,33 between 5% and 20% of total cells or different classes of progenitors contained in the graft were detected in the BM of recipient animals shortly AT, studies examining homing of human cells to the marrow of NOD/SCID recipients11,34 reported substantially lower recoveries. Although data describing recovery of total cells as those we report here are limited,11 recovery of specific classes of progenitor cells has been examined more closely.11,34 van Hennik et al11 reported that only 2.3% of cord blood (CB) CD34+ xenografts could be detected in the marrow of NOD/SCID recipients and demonstrated that, on average, the seeding efficiency of CB progenitors was higher than that of BM and MPB.11 Assuming similar seeding efficiencies between total cells and assayable progenitors, it is plausible that fewer than 2.3% of transplanted BM and MPB CD34+ cells home to the BM of NOD/SCID recipients, a figure in agreement with the 1% to 1.1% recovery rate reported in the present studies. Numbers of cells homing to the BM were 5- to 10-fold higher than those homing to the spleen, similar to previous observations.11 Given the relative mass of the BM and spleen, a higher percentage of transplanted cells would be expected to home to these tissues if homing was a specific and directed event. In fact, a higher percentage of human cells detected in the BM 40 hours AT were CD34+ compared with those present in the spleen (Figure 7), suggesting a more directed homing of these cells to the BM. Many factors, however, including preferential maintenance of CD34 expression in the BM or down-regulation of CD34 expression in the spleen, may explain these differences. Interestingly, human cells detected in the BM of recipient mice reflected the same phenotypic profile of donor cells as previously reported for prenatally transplanted BM cells in a mouse model.35 A recent report by Cashman and Eaves34 indicated that only 7% and 5% of transplanted human cord blood- and fetal liver-derived competitive repopulating units, respectively, could be detected in the BM of NOD/SCID recipients 24 hours AT, suggesting that approximately 19 of every 20 primitive HPCs do not home specifically to the BM. When more mature progenitors were assessed, only 1 in 50 cells homed to the BM within the first 24 hours AT.34 These and similar results demonstrating that only 4% to 5% of human cord blood cobblestone area-forming cells (and a smaller percentage of these cells derived from BM or MPB) home to the marrow of NOD/SCID recipients11 suggest that homing of primitive HPCs to the BM is nonspecific and/or inefficient. Cell cycle activation and proliferation of syngeneic-transplanted cells has been examined in few previous studies with contradicting results. While some studies documented proliferation of transplanted cells shortly after transplantation,16,19 others reported that most of BM-homed hematopoietic cells remained dormant 24 to 48 hours AT.21,33 Our present studies demonstrate that, in both BM and spleen, transplanted human cells remain mitotically quiescent beyond what is required to induce cell cycle activation in vitro. Our results concerning onset of proliferation of transplanted cells are in agreement with some previous studies21,33 and in conflict with others.16,19 The nature of this discrepancy could not be gleaned from our experimental design, although lack of suitable mitogenic signals in the murine marrow microenvironment was ruled out in studies in which human cytokines were administered before, during, and after transplantation of human cells. Whether persistent mitotic quiescence of transplanted cells is a unique characteristic of human progenitor cells or whether the observed behavior of human HPCs resulted from their lodgment in a xenogeneic microenvironment requires further investigation in more physiologic settings. The precise position in the cell cycle in which transplanted cells are arrested could not be determined given the paucity of cells available for such studies. However, data from Table 1 infer that late G1 is a possibility since progression of S/G2+M cells through mitosis was documented followed by accumulation of these cells in what was collectively measured by PI staining as G0/G1. In addition, cells isolated in G0/G1 (Figure 5) or in G1 (Figure 6) also remained in G0/G1, suggesting that late G1 was the stage in cell cycle in which proliferation of these cells was halted. Under this proposal, cells homing to the BM while in G0 phase of cell cycle remain in G0 and may be immune to this late G1 arrest once their proliferation is initiated. Proliferation inhibition observed in these studies may play an important role in determining the fate of transplanted cells and their eventual contribution to hematopoietic reconstitution. On the basis of apoptosis studies, we believe that proliferation inhibition in the BM of recipient animals may serve as a selection mechanism whereby progression through cell cycle of transplanted cycling cells is halted, leading to their demise, whereas their mitotically quiescent counterparts can remain dormant in the marrow without adverse effects on their survival. This hypothesis may explain why ex vivo-expanded cells, composed predominantly of cycling cells, fail to efficiently engraft conditioned animals and may account for why we previously observed that MPB CD34+ cells traversing in short-term culture from G0 into G1 fail to engraft NOD/SCID recipients.17 Certainly, other changes in the functional capacity of expanded cells, including differences in the degree and stage of maturity of cells composing each fraction, alterations in homing and seeding capacities, and diminution in proliferative potential, may contribute to the loss of hematopoietic potential documented for these cells. It remains to be noted that, because the hematopoietic potential of BM-homed cells was not formally proven, our claim that stem cells homing to the BM of transplant recipients remain quiescent for at least 40 hours AT is not entirely conclusive. However, it is unlikely that true repopulating stem cells home to the marrow later than 40 hours AT as was previously demonstrated by other groups.11,16,32,34 It is thus plausible to conclude that transplanted human hematopoietic cells, possibly including stem cells, as examined in the NOD/SCID xenogeneic transplantation model, do not preferentially home to the BM and remain quiescent for a considerable period of time after transplantation. Although this proliferation inhibition may have no deleterious effects on the engraftment of quiescent cells, it may substantially curtail the hematopoietic potential of cycling cells. These studies serve as a first step in elucidating the fate of transplanted hematopoietic cells shortly after their homing to the BM and provide an experimental model suitable for further investigations in this field.
We thank Susan Rice and Jeffrey Lay for their assistance in all the flow cytometric procedures and Dr Mervin Yoder for his critical review of this manuscript.
Submitted January 18, 2001; accepted October 11, 2001.
Supported by National Institutes of Health grants RO1 HL55716 and RO1 HL62200 to E.F.S. and National Institute of Diabetes and Digestive and Kidney Diseases Center for Excellence in Molecular Hematology grant P50 DK49218.
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: E. F. Srour, Indiana University School of Medicine, 1044 W Walnut St, R4-202, Indianapolis, IN 46202-5121; e-mail: esrour{at}iupui.edu.
1. Sanchez M-J, Holmes A, Miles C, Dzierzak E. Characterization of the first definitive hematopoietic stem cells in the AGM and liver of the mouse embryo. Immunity. 1996;5:513-525[CrossRef][Medline] [Order article via Infotrieve]. 2. Yoder M, Hiatt K, Dutt P, Mukherjee P, Bodine D, Orlic D. Characterization of definitive lymphohematopoietic stem cells in the day 9 murine yolk sac. Immunity. 1997;7:335-344[CrossRef][Medline] [Order article via Infotrieve]. 3. Delassus S, Cumano A. Circulation of hematopoietic progenitors in the mouse embryo. Immunity. 1996;4:97-106[CrossRef][Medline] [Order article via Infotrieve]. 4. Tavassoli M. Embryonic and fetal hemopoiesis: an overview. Blood Cells. 1991;1:269-281.
5.
Papayannopoulou T, Nakamoto B.
Peripheralization of hemopoietic progenitors in primates treated with anti-VLA4 integrin.
Proc Natl Acad Sci U S A.
1993;90:9374-9378 6. Hardy CL. The homing of hematopoietic stem cells to the bone marrow. Am J Med Sci. 1995;309:260-266[Medline] [Order article via Infotrieve].
7.
Dercksen MW, Gerritsen WR, Rodenhuis S, et al.
Expression of adhesion molecules on CD34+ cells: CD34+ L-selectin+ cells predict a rapid platelet recovery after peripheral blood stem cell transplantation.
Blood.
1995;85:3313-3319
8.
Jacobsen K, Kravitz J, Kincade PW, Osmond DG.
Adhesion receptors on bone marrow stromal cells: in vivo expression of vascular cell adhesion molecule-1 by reticular cells and sinusoidal endothelium in normal and gamma-irradiated mice.
Blood.
1996;87:73-82 9. Papayannopoulou T, Craddock C. Homing and trafficking of hematopoietic progenitor cells. Acta Haematol. 1997;97:97-104[Medline] [Order article via Infotrieve]. 10. Prosper F, Stroncek D, McCarthy JB, Verfaillie CM. Mobilization and homing of peripheral blood progenitors is related to reversible downregulation of alpha4-beta1 integrin expression and function. J Clin Invest. 1998;101:2456-2467[Medline] [Order article via Infotrieve].
11.
van Hennik PB, de Koning AE, Ploemacher RE.
Seeding efficiency of primitive human hematopoietic cells in nonobese diabetic/severe combined immune deficiency mice: implications for stem cell frequency assessment.
Blood.
1999;94:3055-3061 12. Cui JS, Wahl RL, Shen TL, et al. Bone marrow cell trafficking following intravenous administration. Br J Haematol. 1999;107:895-902[CrossRef][Medline] [Order article via Infotrieve].
13.
Kollet O, Peled A, Lapidot T.
Exclusive homing of human CD38 14. van der Loo JCM, Xiao XL, McMillin D, Hashino K, Kato I, Williams DA. VLA-5 is expressed by mouse and human long-term repopulating hematopoietic cells and mediates adhesion to extracellular matrix protein fibronectin. J Clin Invest. 1998;102:1051-1061[Medline] [Order article via Infotrieve].
15.
Orschell-Traycoff CM, Hiatt K, Dagher RN, Rice S, Yoder M, Srour EF.
Homing and engraftment potential of Sca-1+lin
16.
Nilsson SK, Dooner MS, Quesenberry PJ.
Synchronized cell-cycle induction of engrafting long-term repopulating stem cells.
Blood.
1997;90:4646-4650
17.
Gothot A, van der Loo JCM, Clapp DW, Srour EF.
Cell cycle-related changes in repopulating capacity of human mobilized peripheral blood CD34+ cells in non-obese diabetic/severe combined immune-deficient mice.
Blood.
1998;92:2641-2649
18.
Zanjani ED, Flake AW, Almeida-Porada G, Tran N, Papayannopoulou T.
Homing of human cells in the fetal sheep model: modulation by antibodies activating or inhibiting very late activation antigen-4-dependent function.
Blood.
1999;94:2515-2522 19. Hendrikx PJ, Martens ACM, Hagenbeek A, Keij JF, Visser JWM. Homing of fluorescently labeled murine hematopoietic stem cells. Exp Hematol. 1996;24:129-140[Medline] [Order article via Infotrieve].
20.
Szilvassy SJ, Bass MJ, Van Zant G, Grimes B.
Organ-selective homing defines engraftment kinetics of murine hematopoietic stem cells and is compromised by ex vivo expansion.
Blood.
1999;93:1557-1566
21.
Lanzkron SM, Collector MI, Sharkis SJ.
Hematopoietic stem cell tracking in vivo: a comparison of short-term and long-term repopulating cells.
Blood.
1999;93:1916-1921
22.
Gothot A, Pyatt R, McMahel J, Rice S, Srour EF.
Functional heterogeneity of human CD34+ cells isolated in subcompartments of the G0/G1 phase of the cell cycle.
Blood.
1997;90:4384-4393 23. Shultz LD, Schweitzer PA, Christianson SW, et al. Multiple defects in innate and adaptive immunologic function in NOD/LtSz-scid mice. J Immunol. 1995;154:180-191[Abstract]. 24. Boggs DR. The total marrow mass of the mouse: a simplified method of measurement. Am J Hematol. 1984;16:277-286[Medline] [Order article via Infotrieve].
25.
Srour EF, Brandt JE, Leemhuis T, Ballas CB, Hoffman R.
Relationship between cytokine-dependent cell cycle progression and MHC class II antigen expression by human CD34+ HLA-DR 26. Srour E, Bregni M, Traycoff C, et al. Long-term hematopoietic culture-initiating cells are more abundant in mobilized peripheral blood grafts than in bone marrow but have a more limited ex vivo expansion potential. Blood Cells Mol Dis. 1996;22:68-81[CrossRef][Medline] [Order article via Infotrieve]. 27. Jordan CT, Yamasaki G, Minamoto D. High-resolution cell cycle analysis of defined phenotypic subsets within primitive human hematopoietic cell populations. Exp Hematol. 1996;24:1347-1352[Medline] [Order article via Infotrieve].
28.
Uchida N, He D, Friera A, et al.
The unexpected G0/G1 cell cycle status of mobilized hematopoietic stem cells from peripheral blood.
Blood.
1997;89:465-472 29. Gothot A, Pyatt R, McMahel J, Rice S, Srour EF. Assessment of proliferative and colony-forming capacity after successive in vitro divisions of single human CD34+ cells initially isolated in G0. Exp Hematol. 1998;26:562-570[Medline] [Order article via Infotrieve]. 30. Osawa M, Hanada K, Hamada H, Nakauchi H. Long-term lymphohematopoietic reconstitution by a single CD34-low/negative hematopoietic stem cell. Science. 1996;273:242-245[Abstract].
31.
Spangrude G, Brooks D, Tumas D.
Long-term repopulation of irradiated mice with limiting numbers of purified hematopoietic stem cells: in vivo expansion of stem cell phenotype but not function.
Blood.
1995;85:1006-1016
32.
van der Loo JCM, Ploemacher RE.
Marrow- and spleen-seeding efficiencies of all murine hematopoietic stem cell subsets are decreased by preincubation with hematopoietic growth factors.
Blood.
1995;85:2598-2606 33. Oostendorp RAJ, Ghaffari S, Eaves CJ. Kinetics of in vivo homing and recruitment into cycle of hematopoietic cells are organ-specific but CD44-independent. Bone Marrow Transplant. 2000;26:559-566[CrossRef][Medline] [Order article via Infotrieve].
34.
Cashman JD, Eaves CJ.
High marrow seeding efficiency of human lymphomyeloid repopulating cells in irradiated NOD/SCID mice.
Blood.
2000;96:3979-3981
35.
Shaaban AF, Kim HB, Milner R, Flake AW.
A kinetic model for the homing and migration of prenatally transplanted marrow.
Blood.
1999;94:3251-3257
© 2002 by The American Society of Hematology.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
|
 |
|
  J. Hoggatt, P. Singh, J. Sampath, and L. M. Pelus Prostaglandin E2 enhances hematopoietic stem cell homing, survival, and proliferation Blood, May 28, 2009; 113(22): 5444 - 5455. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. A. Poteat, B. R. Chitteti, K. E. Pollok, A. Suvannasankha, and E. F. Srour Differential Maintenance of Cell Cycle Status of Human Bone Marrow- and Umbilical Cord Blood-Derived CD34+ Cells after Homing to the Bone Marrow Microenvironment. Blood (ASH Annual Meeting Abstracts), November 16, 2008; 112(11): 1327 - 1327. [Abstract]
|
|
|
|
|
|
H. Bonig, A. Wundes, K.-H. Chang, S. Lucas, and T. Papayannopoulou Increased numbers of circulating hematopoietic stem/progenitor cells are chronically maintained in patients treated with the CD49d blocking antibody natalizumab Blood, April 1, 2008; 111(7): 3439 - 3441. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Si, S. Ciccone, F.-C. Yang, J. Yuan, D. Zeng, S. Chen, H. J. van de Vrugt, J. Critser, F. Arwert, L. S. Haneline, et al. Continuous in vivo infusion of interferon-gamma (IFN-{gamma}) enhances engraftment of syngeneic wild-type cells in Fanca-/- and Fancg-/- mice Blood, December 15, 2006; 108(13): 4283 - 4287. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 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]
|
|
|
|
 |
|
 R. W. Garrett and T. A. Gasiewicz The Aryl Hydrocarbon Receptor Agonist 2,3,7,8-Tetrachlorodibenzo-p-dioxin Alters the Circadian Rhythms, Quiescence, and Expression of Clock Genes in Murine Hematopoietic Stem and Progenitor Cells Mol. Pharmacol., June 1, 2006; 69(6): 2076 - 2083. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Verhoeyen, M. Wiznerowicz, D. Olivier, B. Izac, D. Trono, A. Dubart-Kupperschmitt, and F.-L. Cosset Novel lentiviral vectors displaying "early-acting cytokines" selectively promote survival and transduction of NOD/SCID repopulating human hematopoietic stem cells Blood, November 15, 2005; 106(10): 3386 - 3395. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Piccoli, R. Ria, R. Scrima, O. Cela, A. D'Aprile, D. Boffoli, F. Falzetti, A. Tabilio, and N. Capitanio Characterization of Mitochondrial and Extra-mitochondrial Oxygen Consuming Reactions in Human Hematopoietic Stem Cells: NOVEL EVIDENCE OF THE OCCURRENCE OF NAD(P)H OXIDASE ACTIVITY J. Biol. Chem., July 15, 2005; 280(28): 26467 - 26476. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Wang, P. Menendez, F. Shojaei, L. Li, F. Mazurier, J. E. Dick, C. Cerdan, K. Levac, and M. Bhatia Generation of hematopoietic repopulating cells from human embryonic stem cells independent of ectopic HOXB4 expression J. Exp. Med., May 16, 2005; 201(10): 1603 - 1614. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. E. Broxmeyer, C. M. Orschell, D. W. Clapp, G. Hangoc, S. Cooper, P. A. Plett, W. C. Liles, X. Li, B. Graham-Evans, T. B. Campbell, et al. Rapid mobilization of murine and human hematopoietic stem and progenitor cells with AMD3100, a CXCR4 antagonist J. Exp. Med., April 18, 2005; 201(8): 1307 - 1318. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. J. Lawrence Stem cells and the Heisenberg uncertainty principle Blood, August 1, 2004; 104(3): 597 - 598. [Full Text] [PDF]
|
|
|
|
|
|
C. Orelio, K. N. Harvey, C. Miles, R. A. J. Oostendorp, K. van der Horn, and E. Dzierzak The role of apoptosis in the development of AGM hematopoietic stem cells revealed by Bcl-2 overexpression Blood, June 1, 2004; 103(11): 4084 - 4092. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
|
|
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]
|
|
|
|
|
|
T. Yahata, K. Ando, T. Sato, H. Miyatake, Y. Nakamura, Y. Muguruma, S. Kato, and T. Hotta A highly sensitive strategy for SCID-repopulating cell assay by direct injection of primitive human hematopoietic cells into NOD/SCID mice bone marrow Blood, April 15, 2003; 101(8): 2905 - 2913. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Baum, J. Dullmann, Z. Li, B. Fehse, J. Meyer, D. A. Williams, and C. von Kalle Side effects of retroviral gene transfer into hematopoietic stem cells Blood, March 15, 2003; 101(6): 2099 - 2113. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. A. Plett, S. M. Frankovitz, and C. M. Orschell-Traycoff In vivo trafficking, cell cycle activity, and engraftment potential of phenotypically defined primitive hematopoietic cells after transplantation into irradiated or nonirradiated recipients Blood, November 15, 2002; 100(10): 3545 - 3552. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Huygen, O. Giet, V. Artisien, I. Di Stefano, Y. Beguin, and A. Gothot Adhesion of synchronized human hematopoietic progenitor cells to fibronectin and vascular cell adhesion molecule-1 fluctuates reversibly during cell cycle transit in ex vivo culture Blood, September 26, 2002; 100(8): 2744 - 2752. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Wilpshaar, M. Bhatia, H. H. H. Kanhai, R. Breese, D. K. Heilman, C. S. Johnson, J. H. F. Falkenburg, and E. F. Srour Engraftment potential of human fetal hematopoietic cells in NOD/SCID mice is not restricted to mitotically quiescent cells Blood, June 17, 2002; 100(1): 120 - 127. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|||||||
 |
 |
 |
 |
 |
 |
 |
 |
||
| Copyright © 2002 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
Top
Abstract
Introduction
