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HEMATOPOIESIS
From the Departments of Hematology and Obstetrics,
Leiden University Medical Center, The Netherlands; Developmental Stem
Cell Biology, Robarts Research Institute, London, Ontario, Canada; and
Department of Pediatrics/Herman B. Wells Center for Pediatric Research,
Department of Biostatistics/Division of Hematology/Oncology, Department
of Medicine, and Department of Microbiology/Immunology, Indiana
University School of Medicine, Indianapolis, IN.
During fetal development, there is a continued demand for large
numbers of primitive and mature hematopoietic cells. This demand may
require that all potential hematopoietic stem cells (HSCs) migrate
effectively to emerging hematopoietic sites and subsequently contribute
to blood cell production, regardless of their cell cycle status. We
recently established that umbilical cord blood cells in the
G1 phase of the cell cycle have a repopulating potential
similar to cells in G0, suggesting that cycling prenatal and neonatal HSCs may have the same functional capabilities described for quiescent, but not cycling, cells from adult sources. To establish the relationship between cell cycle status and hematopoietic potential at early stages of human ontogeny, the in vivo engraftment potential of
mitotically defined fetal liver (FL) and fetal bone marrow (FBM) cells
were examined in NOD/SCID recipients. Following transplantation of the
same numbers of G0, G1, or S/G2+M
CD34+ cells from FL, equivalent percentages of recipient
mice were chimeric (55%, 60%, and 60%, respectively). FBM-derived
CD34+ cells in all phases of the cell cycle engrafted in
conditioned recipients and sustained human hematopoiesis, albeit at
lower levels than their FL-derived counterparts. Multilineage
differentiation was evident in all transplanted mice independent of the
source or cell cycle status of graft cells. In addition, levels of
chimerism in mice transplanted with fetal blood-derived G0
or G1 CD34+ lineage-depleted cells were
similar. These results support the assertion that mitotically quiescent
and cycling fetal hematopoietic cells contain marrow-repopulating stem
cells capable of multilineage engraftment in NOD/SCID mouse recipients.
(Blood. 2002;100:120-127) During embryonic and fetal life, various tissues
sequentially lose and gain hematopoietic function. It has been
suggested that hematopoietic stem cells (HSCs) are derived from the
same ancestor cells early in development, which give rise to several different extra-embryonic (yolk sac) and embryonic tissues (embryonic mesoderm).1,2 During ontogeny, HSCs may migrate between
these tissues, beginning in the yolk sac and the ventral wall of the dorsal aorta, followed by trafficking to the fetal liver (FL) and
subsequently to fetal bone marrow (FBM).3,4 HSCs are found
in the fetal blood (FB) from 5 weeks of gestation5 onward and are found transiently in the ventral wall of the dorsal aorta before hepatic hematopoiesis occurs.6 Hematopoiesis starts at 6 weeks of gestation in the FL7 and from 14 weeks of
gestation in the FBM8; the latter continues to be the
primary site of hematopoiesis throughout prenatal and adult life.
Unlike peripheral blood of adults, FB contains high concentrations of
hematopoietic progenitors. Because it is accessible by intrauterine
transfusion, FB may be a reasonable source during pregnancy for gene
therapy intervention. Using a murine xenogeneic transplantation model,
we recently established that at 12 to 18 weeks of gestation,
circulating FB is a rich source of NOD/SCID repopulating cells and that
these cells are intrinsically distinct from their counterparts in other
fetal and postnatal sources of HSC.9 Not only do fetal
tissues contain high frequencies of hematopoietic progenitor cells,
they are also a rich source of putative HSCs.10-12 The
percentage of CD34+ cells in FB at 12 to 18 weeks of
gestation is as high as that seen in umbilical cord blood (UCB),
whereas that of CD34+CD38 Adult mobilized peripheral blood (MPB) hematopoietic stem cells capable
of engrafting NOD/SCID mice are predominantly found in the
G0 phase of the cell cycle13 and exhibit a
16-fold enrichment in their repopulating potential compared to their
counterparts residing in G1.13 We recently
reported14 that unlike MPB, the repopulating capacity of
mitotically active and resting cord blood CD34+ cells in
NOD/SCID mice is similar.14 During ontogeny, normal hematopoietic development requires a considerable proliferative output
from HSCs. It is possible that the continuous demand for rapidly
increasing numbers of hematopoietic cells during mammalian development
affords mitotically active HSCs in prenatal and neonatal tissue
engraftment and hematopoietic potentials that are restricted to
mitotically quiescent cells in adult tissues.13,14 In the context of gene transfer protocols, these mitotically active, yet
engrafting, cells might be of great importance because of the need for
cell cycle activation and progression through the cell cycle before
efficient gene integration can be achieved in target cells. Fetal
circulating blood hematopoietic progenitor cells have been shown to be
more susceptible to retrovirus-mediated gene transfer than cells from
adult tissues, most likely because of their mitotically active
state.15 To investigate the relationship between cell
cycle status and hematopoietic potential during the early stages of
human ontogeny and to provide a static assessment of the distribution
of engrafting stem cells across phases of the cell cycle, the
engraftment capacity of mitotically defined fractions of fetal
hematopoietic precursor cells was examined in NOD/SCID mice. In this
report, we demonstrate that unlike adult mobilized peripheral blood and
BM cells, but analogous to neonatal cord blood cells, fetal
hematopoietic progenitors from blood, liver, and bone marrow residing
in quiescent and active phases of the cell cycle contain putative stem
cells capable of multilineage and sustained engraftment in NOD/SCID
mouse recipients.
Collection and purification of fetal liver and bone marrow
CD34+ cells
Collection and isolation of lineage-depleted CD34+
fetal peripheral blood cells
Cell cycle fractionation with Hoechst 33342 and Pyronin Y To distinguish between FL, FBM, and FB progenitor cells in the G0 or G1 phase of the cell cycle, which have similar DNA but different RNA content, and between G0/G1 and S/G2+M cells, which have different DNA and RNA content, simultaneous DNA and RNA staining with Hoechst 33342 (Hst) and pyronin Y (PY), respectively, was performed as previously described16,17 (Figure 1). Briefly, purified progenitor cells were resuspended in 1 µg/mL Hst (Molecular Probes, Eugene, OR) prepared in Hst buffer consisting of Hanks balanced salt solution (BioWhittaker, Walkersville, MD), 20 mM HEPES (BioWhittaker), 1 g/L glucose, 10% fetal calf serum (FCS; Hyclone, Logan, UT), and verapamil at 50 µM (Sigma, St Louis, MO). After 45-minute incubation at 37°C, PY (Polysciences, Warrington, PA) was added at a final concentration of 1 µg/mL. Cells were further incubated for another 45 minutes at 37°C, then washed once in chilled Hst buffer. FL or FBM CD34+ cells were incubated for 30 minutes with fluorescein isothiocyanate (FITC)-conjugated anti-CD34 monoclonal antibodies (PharMingen, San Diego, CA) at 4°C. Cells were washed again, resuspended in Hst buffer, and analyzed or sorted on a FACStar Plus (Becton Dickinson Immunocytometry Systems [BDIS], San Jose, CA). Gated FL and FBM CD34 FITC-positive cells were identified as in G0/G1 or S/G2+M based on their Hst staining profile. Cells in G0/G1were subdivided into those in G0 based on their minimal RNA content, whereas cells traversing into G1 were defined as those with high or maximal RNA staining.17 Cells in S/G2+M have higher than 2n DNA (high Hst) and high RNA (high PY) content. Fetal blood CD34+Lin
cells were first stained with Hst and PY and subsequently with allophycocyanin-conjugated CD34 monoclonal antibodies (BDIS). Cells were sorted on a Vantage SE (BDIS) to isolate G0 and
G1 cells within the CD34+Lin cell
fraction. Sorting gates were designed as previously
reported.13,18 This staining protocol allowed the
isolation of viable FL and FBM CD34+ cells in
G0 (G0CD34+), G1
(G1CD34+), or S/G2+M
(S/G2+MCD34+), and viable FB
CD34+Lin cells in G0
(G0CD34+Lin) or G1
(G1CD34+Lin). During sorting,
cells were kept on ice to minimize dye leaking and were protected from
light. Viability of sorted cells always exceeded 95%. Validation of
cell cycle status of sorted cells was performed using propidium iodide
staining (Figure 1).
In vitro analysis of hematopoietic progenitor cells To assay FL and FBM CD34+ cells for their original content of clonogenic hematopoietic progenitors, G0CD34+, G1CD34+, and S/G2+MCD34+ cells from one fetus were separated by cell sorting. For these assays, sorted G0, G1, or S/G2+MCD34+ cells were assayed in duplicate in plastic 35-mm tissue culture dishes in 1 mL IMDM containing, at the final concentration, 30% FCS, 1.3% methylcellulose, 5 × 105 M 2-mercaptoethanol (Sigma),
50 ng/mL stem cell factor, 10 ng/mL interleukin-3 (IL-3), 10 ng/mL
interleukin-6 (IL-6), 5 ng/mL granulocyte macrophage-colony-stimulating factor (GM-CSF), and 2 U/mL
erythropoietin. All cytokines were kind gifts from Amgen (Thousand
Oaks, CA). Plates were cultured at 37°C in 100% humidified
atmosphere containing 5% C02. After 2 weeks, plates were
scored by means of an inverted microscope. To estimate the frequencies
of long-term culture initiating cells (LTC-ICs) in the various
fractions, LDA assays were performed as previously
described19 with some modifications.20 M2-10B4 cells were irradiated at 80 Gy (GammaCell 40; Nordion
International, Kanata, Ontario, Canada) and were plated in
flat-bottom 96-well plates at a concentration of
15 × 103 cells/well in 100 µL long-term culture medium
(LTCM). LTCM consisted of Myelocult (Stem Cell Technologies, Vancouver,
BC, Canada) containing 106 mM hydrocortisone (Sigma).
After 24 hours, test cells in 100 µL LTCM were added to the plated
stromal cells in limiting dilution (64 to 8 cells per well, in a 2-fold
dilution scheme using 48 wells per cell dose). Plates were maintained
at 37°C in 100% humidified atmosphere containing 5%
CO2, with weekly half-medium changes. After 5 weeks, 120 µL medium was removed from each well, followed by the addition of 150 µL IMDM containing methylcellulose and growth factors as described
above. After 2 weeks, wells were scored for the presence or absence of
hematopoietic colonies, and the frequency of LTC-IC was calculated
using the maximum likelihood estimator.21
Transplantation of test cells into NOD/SCID mice NOD/LtSz-scid/scid (NOD/SCID) mice22 used in these experiments were bred and housed at Indiana University or at the Robarts Research Institute. Mice used at Indiana University were kindly provided by Dr D. A. Williams (Indianapolis, IN). Animal experiments were performed in accordance with institutional guidelines approved by the Animal Care Committee of the Indiana University School of Medicine and the Robarts Research Institute/University of Western Ontario. Nine- to 12-week old NOD/SCID mice were sublethally irradiated with 3 Gy from a cesium Cs 137 source (GammaCell 40). Mice received by intravenous injection 107 nonadherent CD34
adult BM cells irradiated with 80 Gy as accessory cells. Two hours
later, mice received transplants of 3.4 × 104 to
6 × 105 G0CD34+,
G1CD34+, or S/G2+MCD34+
cells from FL or FBM or
G0CD34+Lin or
G1CD34+Lin cells from FB. FL and
FBM were obtained from the same fetus, and equal numbers of each group
of cells were transplanted into each recipient. FB cells were obtained
from different fetuses, and equal numbers of
G0CD34+Lin or
G1CD34+Lin cells were
transplanted. After 8 weeks, blood was collected, the mice were killed
by cervical dislocation, and the spleens and bone marrow were
harvested. Bone marrow, spleen, and peripheral blood were analyzed for
chimerism in recipients of FL and FBM grafts, whereas only the BM of
mice that received FB transplants were analyzed for engraftment.
Flow cytometric analysis of engraftment The level of chimerism in recipient mice was determined by flow cytometric assessment of the percentage of CD45+ cells contained in these animals. Single-cell suspensions of BM, spleen or blood were incubated with FITC-conjugated mouse anti-human CD45 or isotype control monoclonal antibodies (PharMingen) for 20 minutes at 4°C. Samples were analyzed on a FACScan (BDIS). Positive cells were identified by comparison with isotypic controls and with cells harvested from control (not those receiving transplants) NOD/SCID mice stained with the same antibodies. To determine the frequencies of subsets of human cells, BM and spleen cell suspensions, which contained more than 1% CD45+ cells, were also stained with phycoerythrin-conjugated CD19, CD20, CD33, CD34, and CD38 or FITC-conjugated CD61, CD3, CD4, and CD8 in different combinations and with Cy-5-conjugated anti-CD45 antibody (PharMingen) as the third color.Progenitor cell analysis of mouse bone marrow To enumerate different HPCs contained in the BM of mice 8 weeks after transplantation, murine BM cell suspensions that contained more than 1% human CD45+ cells were analyzed in progenitor cell assays, allowing preferential colony formation of human precursor cells. Total cell suspensions (between 1.1 × 104 and 7.5 × 104 total cells) containing 2 × 103 CD45+CD34+ human cells (as determined by flow cytometric analysis of these samples) were assayed in duplicate in plastic 35-mm tissue culture dishes in 1 mL IMDM containing, at final concentration, 1.3% methylcellulose, 30% FCS, 5 × 105 M 2-mercaptoethanol, 50 ng/mL stem cell
factor, 10 ng/mL IL-3, 10 ng/mL IL-6, 5 ng/mL GM-CSF, and 2 U/mL
erythropoietin. Cultures were incubated at 37°C in 100% humidified
atmosphere containing 5% CO2. Hematopoietic colonies were
scored after 2 weeks using an inverted microscope. Only 2 granulocyte
macrophage-colony-forming unit (CFU-GM)-derived colonies were
detected when 4 × 104 control murine BM cells were
plated under these conditions (n = 2).
Statistical analysis A general linear model procedure (analysis of variance) was used to examine the association between percentage chimerism and position of cells in the cell cycle after adjusting the number of cells infused. The interaction between the cell cycle status of transplanted cells and the number of cells infused was also examined to assess whether the effect of cells infused on chimerism was similar between cells in different positions in the cell cycle. Least-squares means of chimerism are reported after they were adjusted for number of cells infused. Where applicable, mean ± SD or mean ± SEM of multiple measurements is reported. Data were analyzed using a Student t test, and differences yielding P < .05 were considered statistically significant.
Cell cycle fractionation of FL and FBM CD34+ cells
and CD34+Lin cells than FBM cells. In addition,
FBM CD34+ cells were uniformly bright for the expression of
CD38, whereas 2 distinct populations of
CD34+CD38bright and
CD34+CD38dim were identified among FL
CD34+ cells. Cell cycle distribution of FL and FBM
CD34+ cells revealed that the percentages of cells residing
in the different phases of cell cycle were comparable in both tissues (Figure 1; Table 1).
Progenitor cell content of FL and FBM G0CD34+, G1CD34+, and S/G2+M CD34+ cells Whether fractionation of FL and FBM CD34+ cells based on their position in cell cycle results in the compartmentalization of HPCs was first examined in vitro. The numbers of clonogenic progenitor cells contained in 103 G0, G1, and S/G2+M fractions of FL and FBM CD34+ cells are shown in Table 1. Similar frequencies of colony-forming cells were detected in all 3 fractions of fetal BM cells. Relatively higher frequencies of progenitors were detected in G1 and S/G2+M fractions of FL CD34+ cells than in G0CD34+ cells. LTC-ICs were detected in the G0, G1, and S/G2+M fractions of CD34+ cells from FL and FBM (Table 1). Both G0 and G1 fractions of FB CD34+Lin cells contained comparable
frequencies of assayable progenitors (Table 1). These results indicated
that equivalent frequencies of primitive hematopoietic cells were
distributed among all phases of the cell cycle in these tissues and
that cell cycle fractionation of FBM and FL CD34+ cells may
not lead to efficient sequestration of HSCs. It is important to point
out, however, that given the difference in distribution of cells among
different phases of the cell cycle (Figure 1), the relative absolute
numbers of progenitors in each compartment were different (Table
1).
NOD/SCID repopulating ability of FL, FBM, and FB CD34+ cells isolated in different phases of the cell cycle The marrow-repopulating ability of G0, G1, and S/G2+MCD34+ cell populations from pooled (1-6 samples per experiment) cryopreserved FL and FBM samples and of G0 and G1CD34+Lin
cells from FB was assessed by transplanting test cells into conditioned NOD/SCID recipients. To confirm that verapamil had no adverse effects
on the functional properties of test cells, the levels of chimerism in
NOD/SCID mice transplanted with verapamil-treated and untreated sorted
FB cells was assessed in a pilot study. In 2 experiments, chimerism
rates in recipients of FB
G0CD34+Lin cells treated with
verapamil were 12% and 12%, whereas those in recipients of untreated
G0CD34+Lin cells were 14% and
10%. Chimerism rates in recipients of verapamil-treated G1CD34+Lin cells were 8% and
4%, and in mice receiving untreated cells they were 6% and 4.5%.
Lack of adverse effects of cryopreservation on the isolation of
engrafting cells in different phases of the cell cycle was also
investigated in a pilot study using FB cells. Freshly isolated
G0CD34+Lin and
G1CD34+Lin cells promoted
12% ± 3% and 7% ± 3% (n = 4) chimerism, respectively. Cells
isolated from the same samples after remaining cryopreserved for 1 week
supported 14% ± 5% and 6% ± 3% chimerism for
G0CD34+Lin and
G1CD34+Lin cells, respectively.
In 7 separate experiments, a total of 26 mice received transplants of
FL CD34+ cells, and 26 mice receiving transplants of FBM
CD34+ cells were separated into G0,
G1, or S/G2+M using between
3.4 × 104 and 6 × 105 cells per animal.
Similarly, equal numbers of G0 or
G1CD34+Lin
Similar percentages of recipient mice were chimeric following
transplantation of FL G0, G1, or
S/G2+MCD34+ cells (55%, 60%, and 60% of mice
that received transplants contained 1% or more CD45+ cells
in the marrow, respectively; Table 2). A
more variable distribution of chimeric animals with 1% or more human
CD45+ cells in BM was observed among recipients of FBM
cells in different phases of the cell cycle (Table 2). When lower
levels of engraftment (more than 0.1% CD45+) were included
in the analysis, comparable numbers of engrafted animals were found
(Table 2).
When G0 and G1
CD34+Lin Differentiation potential of repopulating FL and FBM cells To compare the in vivo differentiation potential of total engrafting cells, the phenotypic profile of chimeric CD45+ cells in bone marrow of recipient mice, resulting from the composite differentiation of all engrafting cells, was determined by 3-color immunostaining. Data generated from these analyses are shown in Figure 3. A large fraction of human CD45+ cells in recipients of FL cells was also CD34+ (24% ± 14%, 20% ± 11%, and 17% ± 0.5% for cells in G0, G1, and S/G2+M, respectively). Among chimeric FL-derived cells, CD19 expression (58% ± 20% for G0 cells, 51% ± 16% for G1 cells, and 29% ± 6% for S/G2+M cells, as percentages of total CD45+ cells) and CD33 expression (27% ± 13% for G0 cells, 28% ± 7% for G1 cells, and 34% ± 15% for S/G2+M cells as percentages of total CD45+ cells) was identifiable, demonstrating that repopulating cells in all phases of the cell cycle were capable of lymphoid and myeloid differentiation. Although smaller numbers of chimeric mice could be analyzed after transplantation with FBM cells because of the lower engraftment levels, all chimeric mice contained CD45+ cells expressing CD34 and CD19 or CD33 cells, illustrating again that FBM-repopulating cells in all phases of the cell cycle were capable of multilineage differentiation. However, given the small numbers of mice that received transplants analyzed in some cases (FL and FBM S/G2+M cells and FBM G1 cells), it is difficult to ascertain that no significant differences in the ability of these cells to sustain multilineage differentiation can be concluded. Phenotypic analysis of engrafting cells in recipients of FB grafts also documented multilineage differentiation capacity among recipients of G0 and G1 CD34+Lin cells (data not shown).
Human progenitor cells in the marrow of recipient NOD/SCID mice To compare the different classes of progenitors in bone marrow of recipient mice at 8 weeks after transplantation, BM cell suspensions from recipients demonstrating 1% or more CD45+ chimerism were assessed in progenitor cell assays (Figure 4). The number of clonogenic progenitors contained in 2 × 103 CD45+CD34+ cells derived from mice that received transplants of FL G0CD34+ cells was 110 ± 30, whereas that detected in an equivalent number of bone marrow cells from G1CD34+ recipients was 71 ± 22 and from S/G2+MCD34+ recipients it was 7 ± 2 (Figure 4). The difference in the number of clonogenic progenitors contained in recipients of FL G0CD34+ cells and recipients of G1CD34+ cells did not reach statistical significance. In contrast to FL recipients, mice that received transplants of cycling FBM cells contained higher numbers of clonogenic cells than recipients of quiescent cells from the same source. Although 240 ± 52 and 250 ± 47 colonies were detected in the marrow of FBM S/G2+M and G1CD34+ recipients, respectively, only 140 ± 42 colonies were present in the BM of mice that received transplants of FBM G0CD34+ cells. However, these differences were not statistically significant.
Mathematical model for engraftment of human cells in NOD/SCID recipients The relationship between the number of transplanted FL and FBM cells and the level of chimerism detected 8 weeks later was analyzed using a general linear models procedure, as previously applied to the analysis of the engraftment of umbilical cord blood cells in G0 or G1 in NOD/SCID mice.14 The relationship between the position of cells in the cell cycle and the number of cells infused can be analyzed using the equation: Y = +  1 X1 + 2 X2 + 3 X3,
where Y is the estimated level of chimerism in
transplanted mice, X1 is an indicator variable
equal to 1 if G1 cells are considered (zero otherwise),
X2 is another indicator variable equal to 1 if
S/G2 +M is considered (zero otherwise), and
X3 is the number of cells infused in thousands.
A constant derived from the linear model, represents the
Y intercept. The value of 1 represents the
expected difference between the levels of chimerism obtained with
G0 versus G1 cells, whereas 2
represents the expected difference between the levels of chimerism
obtained with G0 versus S/G2 +M cells.
3 is the slope representing the expected incremental
change in chimerism for every additional 103 cells
contained in the graft.
1 = 2 = 3 = 0
represents the null hypothesis that there is no relationship between
level of chimerism and cell cycle status or number of transplanted
cells. If the analysis reaches statistical significance
(P < .05), the hypothesis is rejected, indicating
that a linear relationship exists between chimerism and cell cycle
status or number of cells transplanted.
When FBM or FL cells were transplanted, no significant relationship was
observed between chimerism in BM and either the number of cells infused
or the position of transplanted cells in the cell cycle. These analyses
indicated that fetal BM or FL-derived chimerism in the BM of recipient
mice is independent of the position of graft cells in the cell cycle
and the number of cells transplanted. For the analysis of chimerism
after transplantation with FB cells,
While examining the hematopoietic repopulating potential of umbilical cord blood cells residing in different phases of the cell cycle, we recently14 proposed that the extensive demand for primitive and mature hematopoietic cells in the developing fetus may require that all potential HSCs contribute to blood cell production regardless of their position in the cell cycle. Such a requirement would necessitate that during embryonic development, mitotically quiescent cells and cells in active phases of the cell cycle home and sustain long-term hematopoiesis to support cell production and maintenance of the stem cell pool as hematopoiesis shifts from the yolk sac to the fetal liver and the BM. As a first step in examining this hypothesis, we recently showed that UCB cells in the G1 phase of the cell cycle have a repopulating potential similar to that of cells in G0,14 suggesting that cycling prenatal and neonatal hematopoietic cells may have the same functional capabilities described for quiescent, but not cycling, adult BM and MPB cells.13 In this report, we demonstrate that as documented for UCB-derived repopulating cells,14 HSCs from earlier stages of human hematopoietic ontogeny, such as fetal liver, fetal blood, and fetal bone marrow, can be found in active phases of the cell cycle. These findings further support our contention that the prevailing dogma ascribing primitive hematopoietic functions to cells residing in deep dormancy in adults may not be true for cells derived from fetal tissues. That fetal HPCs are functionally distinct from similar cells from adult tissues has been demonstrated. Lansdorp et al10 demonstrated that FL cells had a higher proliferative capacity than cells derived from CB or adult BM and were capable of generating more CD34+ cells in culture. These authors concluded that the turnover rate and both the proliferative and differentiation potentials of HPCs decrease during ontogeny.10 More recently, it has been demonstrated that FL is enriched for NOD/SCID repopulating cells compared to CB cells.12,23 Under these conditions, the functional capacity assessed by these studies10,12,23 would have been contributed by resting and cycling fetal cells but only by quiescent cells derived from adult tissues. It is intriguing that cells in the S/G2+M phases of the cell cycle from FL and FBM engrafted durably. Our previous studies examining the relationship between in vivo engraftment and the position of graft cells in cell cycle focused mainly on mobilized peripheral blood and UCB, sources of stem cells known to be predominantly in G0 or G1.13,14,24-26 Marrow CD34+ cells contain a small fraction of cells in S/G2+M, and that has precluded testing of these cells in the NOD/SCID model. Based on our results from BM and mobilized peripheral blood studies,13 it is unlikely that cells in S/G2+M from adult sources would engraft in vivo. This prediction, drawn from studies examining the marrow-repopulating potential of murine HSCs in active phases of the cell cycle,27-30 and the general belief that stem cells reside in deep dormancy within the BM microenvironment31,32 are in sharp contrast to our present observations. Progression of cells into active phases of the cell cycle in ex
vivo expansion cultures has long been suspected to be the primary
reason expanded cells exhibit in vivo functions inferior to those of
freshly isolated cells.33-36 However, mechanisms
underlining this defect remain unresolved. In addition, it has been
postulated that HSCs undergoing cell division in vitro may modulate
adhesion molecules important for homing and engraftment, thus losing
their repopulating potential.37-40 Cell cycle-associated
modulation of expression of adhesion molecules by candidate stem cells
has been previously reported.41 It was therefore
speculated that cycling cells in vivo manifest limitations similar to
those documented for cells cycling in vitro (loss of hematopoietic
function) but that though the re-entry of cycling cells into mitotic
quiescence and the reacquisition of function is possible in vivo, it is
not achievable under conditions used for ex vivo expansion of HSCs. In
a recent report, Glimm et al42 documented that ex
vivo-expanded CB cells appear to fail to re-enter G0, as
demonstrated by their inability to reconstitute NOD/SCID mice. Although
in these studies limited numbers of G0 cells recovered from
expansion cultures could be transplanted into immunodeficient mice,
these grafts were large enough to contain sufficient numbers of
engrafting cells (relative to unmanipulated cells) that might have
returned to dormancy. Our present and previous14,20 studies
indicate that whereas cycling stem cells from adult sources might
suffer from in vivo loss of function, HSCs in fetal tissues retain
their functional properties throughout the cell cycle. Whether
mechanisms associated with loss of function of cells from mature
hematopoietic tissues as they traverse the cell cycle are not operative
in fetal cells has not yet been determined. Similarly, whether the
modulation of adhesion molecules does not accompany the movement of
fetal HSCs through the cell cycle or whether modulation of these
markers is minimal or limited to molecules not involved in homing and engraftment remains to be established. Finally, it remains to be
determined whether the repopulating ability observed in cycling fetal
and neonatal cells14 Maintenance of engraftment potential of fetal HSCs as they progress through the cell cycle was also confirmed by analysis of engraftment with a general linear models procedure.14 These analyses indicated that for FL and FBM, no significant relationship was observed between chimerism in the BM and the position of cells in either G0 or G1, indicating that the level of chimerism in these recipients is independent of the position of graft cells in the cell cycle. Although for FB both phases of the cell cycle showed engraftment, the effect of the number of cells infused on chimerism was different, depending on the cell cycle position of transplanted cells. In part, this might have been caused by the low number of cells transplanted or by the fact that a subfraction of CD34+ cells was used in these studies. Recently, we documented that a substantial fraction of adult
human BM cells residing in the S/G2+M phases of the cell cycle undergo
apoptosis shortly after their trafficking to the BM of conditioned
NOD/SCID recipients.45 In these studies,45 a
significantly smaller percentage of cells in
G0/G1 detected in the BM of recipient mice were
apoptotic, suggesting that whereas homing or trafficking of cycling
adult hematopoietic cells may be intact, apoptosis may play a role in
the loss of function of these cells. In view of these results, it would
be interesting to examine whether cycling fetal hematopoietic cells are
more resistant to apoptosis. It is unlikely that engraftment of cycling
cells in these studies was enhanced by the recently described
phenomenon46 attributed to the conditioning of recipient
mice with apoptotic leukocytes (irradiated nonadherent BM
CD34 Results obtained with FB may be of particular interest in view of possible prenatal correction of certain inborn errors. This source of HPCs has not been extensively investigated, but it represents a promising source of stem cells suitable for gene therapy.15 Our transplantation data presented here, and the results of gene transfer studies using FB cells,15 demonstrate that these cells are not only enriched for marrow-reconstituting cells but are also capable of retaining in vivo hematopoietic functions following in vitro manipulations required for effective retroviral-mediated gene transfer. These results are encouraging for 2 reasons. First, they establish the usefulness of FB cells as a source of transplantable stem cells and as targets for effective retroviral-mediated gene transfer. Second, they illustrate that HSCs from fetal tissues may be less susceptible to the observed loss of function associated with ex vivo manipulation of cells from adult sources during transduction. That fetal HSCs traverse the cell cycle with minimal loss of function has been suggested in studies demonstrating the maintenance of SRC function in retrovirally marked clones expressed in NOD/SCID mice.15 However, it is important to stress that the loss of hematopoietic potential after ex vivo manipulation of fetal hematopoietic cells has been shown in earlier studies using different in vitro assays.10,47,48 In conclusion, these studies summarize our assessment of the hematopoietic potential of cells in different phases of the cell cycle throughout ontogeny. Although the engraftment potential of marrow-repopulating cells is restricted to quiescent cells in adult tissues, cycling and resting fetal and neonatal cells retain this functional property. Although the biologic significance of maintenance of engraftment potential in fetal HSCs at all phases of the cell cycle may lie in the continued demand for large numbers of hematopoietic cells and the possible migration of these cells from one site to another during fetal development, the significance of this property in reconstitutional therapies still has to be established.
Submitted July 9, 2001; accepted March 5, 2002.
Supported by National Institutes of Health grant R01 HL55716 (E.F.S.), National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Center for Excellence in Molecular Hematology grant P50 DK49218, and the J. A. Cohen Institute for Radiopathology and Radiation Protection.
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: Edward F. Srour, Indiana University School of Medicine, R4-202, 1044 West Walnut St, Indianapolis, IN 46202-5121; e-mail: esrour{at}iupui.edu.
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Rich IN.
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© 2002 by The American Society of Hematology.
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  S. Knaan-Shanzer, I. van der Velde-van Dijke, M. J.M. van de Watering, P. J. de Leeuw, D. Valerio, D. W. van Bekkum, and A. A.F. de Vries Phenotypic and Functional Reversal Within the Early Human Hematopoietic Compartment Stem Cells, December 1, 2008; 26(12): 3210 - 3217. [Abstract] [Full Text] [PDF]
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F. Hermitte, P. Brunet de la Grange, F. Belloc, V. Praloran, and Z. Ivanovic Very Low O2 Concentration (0.1%) Favors G0 Return of Dividing CD34+ Cells Stem Cells, January 1, 2006; 24(1): 65 - 73. [Abstract] [Full Text] [PDF]
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K. Leibundgut, N. M.R. Schmitz, and A. Hirt Catalytic Activities of G1 Cyclin-Dependent Kinases and Phosphorylation of Retinoblastoma Protein in Mobilized Peripheral Blood CD34+ Hematopoietic Progenitor Cells Stem Cells, August 1, 2005; 23(7): 1002 - 1011. [Abstract] [Full Text] [PDF]
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R. N. La Motte-Mohs, E. Herer, and J. C. Zuniga-Pflucker Induction of T-cell development from human cord blood hematopoietic stem cells by Delta-like 1 in vitro Blood, February 15, 2005; 105(4): 1431 - 1439. [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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Abstract
Introduction
but not their counterparts from adult tissues