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Blood, Vol. 94 No. 9 (November 1), 1999:
pp. 3055-3061
Seeding Efficiency of Primitive Human Hematopoietic Cells in Nonobese
Diabetic/Severe Combined Immune Deficiency Mice: Implications for Stem
Cell Frequency Assessment
By
Paula B. van Hennik,
Alexandra E. de Koning, and
Rob E. Ploemacher
From the Institute of Hematology, Erasmus University Rotterdam,
Rotterdam, The Netherlands.
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ABSTRACT |
Nonobese diabetic/severe combined immune deficiency (NOD/SCID) mouse
repopulating cells (SRC) have been proposed to represent a more
primitive human stem cell subset than the cobblestone area-forming cell
(CAFC) week (wk) 6 or the long-term culture-initiating cell (LTC-IC) wk
5 on the basis of their difference in frequency, phenotype, transfectibility, and multilineage outgrowth potential in
immunodeficient recipients. We have assessed the
percentage of various progenitor cell populations (colony-forming cell
[CFC] and CAFC subsets) contained in unsorted NOD/SCID BM nucleated
cells (nc), human umbilical cord blood (UCB) nc, bone marrow (BM) nc,
peripheral blood stem cells (PBSC), and CD34+ selected
UCB nc, seeding in the BM and spleen of NOD/SCID mice within 24 hours
after transplantation. The seeding efficiency of NOD/SCID BM CAFC wk 5 was median (range) in the spleen 2.9% (0.7% to 4.0%) and in the
total BM 8.7% (2.0% to 9.2%). For human unsorted UCB nc, BM nc,
PBSC, and CD34+ UCB cells, the seeding efficiency for
CAFC wk 6 in the BM of NOD/SCID mice was 4.4% (3.5% to 6.3%), 0.8%
(0.3% to 1.7%), 5.3% (1.4% to 13.6%), and 4.4% (3.5% to 6.3%),
respectively. Using flow cytometry, the percentage CD34+
UCB cells retrieved from the BM of sublethally or supralethally irradiated NOD/SCID mice was 2.3 (1.4 to 2.8) and 2.5 (1.6 to 2.7),
respectively. Because we did not observe any significant differences in
the seeding efficiencies of the various stem cell subsets, it may be
assumed that the SRC seeding efficiency in NOD/SCID mice is similarly
low. Our data indicate that the seeding efficiency of a graft can be of
great influence when assessing stem cell frequencies in in vivo
repopulation assays.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
OVER THE PAST decade various in vitro and
in vivo surrogate assays have been developed to estimate human stem
cell frequencies. The cobblestone area-forming cell
(CAFC)1,2 and long-term culture-initiating cell
(LTC-IC)3 assays have been proposed to determine and
enumerate primitive progenitor cells with long-term repopulating
ability in vitro in both the murine and human hematopoietic system. A
quantitative in vivo assay for human hematopoietic stem cells has been
established by the recent development of the nonobese diabetic/severe
combined immune deficiency (NOD/SCID) mouse model.4 In this
model, human (stem) cells are infused into the tail vein of a
sublethally irradiated NOD/SCID mouse, with optional intraperitoneal
administration of cytokines to facilitate outgrowth of the stem cells
that homed to the bone marrow. Thirty-five days after transplantation,
the bone marrow (BM) cells of the NOD/SCID mice are collected and the
percentage of human chimerism is determined using either flow cytometric analysis of the pan-leukocyte marker CD45 or Southern blot
analysis for the human chromosome 17-specific  -satellite probe.4 Stage- and/or lineage-specific antibodies are used to determine multilineage outgrowth. On the assumption that every infused SRC will generate detectable human engraftment, limiting dilution techniques have been used to establish the frequency of the
SRC in umbilical cord blood (UCB), BM, and peripheral blood stem cells
(PBSC), ie, UCB 1:930,000 nucleated cells (nc); BM 1:3,000,000 nc and
PBSC 1:6,000,000 nc.5 Conneally et al6 obtained
similar results in enumerating the frequency of the competitive repopulating units (CRU) in UCB. However, from syngeneic murine studies, it is known that only 18% to 20% of the infused stem cells
home to the total BM and 8% to 10% lodge in the spleen.7 Additionally, it is still unknown whether all stem cells that reach the
BM actually contribute to hematopoietic reconstitution of depleted
marrow spaces. The cells that do not reach their niches in
hematopoietic tissues are likely to be sequestered in organs with large
capillary beds like the liver and lungs.8 The seeding efficiency of human stem cells in the NOD/SCID mouse could even be
lower than in the syngeneic murine situation. This implicates that the
seeding efficiency of a graft can be of great influence when assessing
stem cell frequencies in repopulation assays. To estimate the seeding
efficiency of human stem cells in the NOD/SCID mouse, we have assessed
the percentages of CAFC subsets and colony-forming cell (CFC) that home
to the BM and spleen of the NOD/SCID mouse within the first 24 hours
postinfusion. Additionally, we have studied the seeding efficiency of
CD34+ selected UCB cells using in vitro culture assays, as
well as flow cytometric analysis.
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MATERIALS AND METHODS |
Human cells.
In the described experiments, cells from different human grafts were
used. The UCB samples were obtained from umbilical cords from
full-term, healthy newborns. The cord blood cells were 1:1 diluted with
Hanks' Balanced Salt Solution (HBSS; GIBCO, Breda, The Netherlands)
and processed using either a Ficoll-gradient (1.077 g/cm2;
Nycomed, Oslo, Norway) or a 3% gelatine solution (Sigma, St Louis,
MO). After harvesting, the mononuclear cell fraction or the
erythrocyte-depleted fraction, respectively, the cells were washed
twice in HBSS. The BM nc were obtained by posterior iliac crest
puncture from 5 hematological healthy adults. The mononuclear cell
fraction was isolated from the BM cells using a Ficoll-gradient as
described above. The PBSC were obtained from 5 patients with non-Hodgkin's lymphoma (NHL) and 1 patient with multiple myeloma (MM).
The mononucleated cell fraction was isolated using a
Ficoll-gradient. These patients were in complete
remission. The patients, the allogeneic donors, and the mothers of the
newborns gave their informed consent. The UCB nc, BM nc, and PBSC were
cryopreserved until use in 10% dimethyl sulfoxide (DMSO; BDH, Poole,
UK) and 20% heat-inactivated fetal calf serum (FCS; Summit
Biotechnology, Fort Collins, CO). The unsorted human stem cells were
used for transplantation into 9 Gy irradiated NOD/SCID mice, CAFC, and
CFC cultures.
NOD/SCID mouse BM cells for transplantation.
Male and female specific pathogen-free NOD/LtSz-scid/scid (NOD/SCID)
mice, 3 to 5 weeks of age, were obtained from the Department of
Immunology at the Erasmus University Rotterdam. The mice were included
in experiments at the age of 6 to 9 weeks. To obtain NOD/SCID BM cells
for transplantation, in total 15 NOD/SCID mice were killed using
CO2 asphyxiation and both femora and tibia were removed.
The NOD/SCID BM cells were harvested using crunching of the bones in
HBSS with 5% FCS. The cells were collected and sieved through a
100-µm sieve. After centrifugation at 1,800 rpm for 8 minutes, the
cells were resuspended in phosphate-buffered saline (PBS; GIBCO). The
NOD/SCID BM cells were used for transplantation into 9 Gy irradiated
NOD/SCID mice, CAFC, and CFC cultures.
Isolation of CD34+ UCB cells.
The mononucleated cell fraction of UCB samples was thawed and using the
Macs-selection system (Miltenyi Biotec GmbH, Bergisch Gladbach,
Germany), CD34+ cells were isolated according to the
manufacturer's instructions. The purity ranged from 72% to 99%. The
CD34+ UCB cells were used for transplantation into 3.5 or 9 Gy irradiated NOD/SCID mice and flow cytometric analysis.
NOD/SCID repopulating cell (SRC) frequency analysis in UCB.
To estimate the frequency of the SRC in UCB, 5 to 21 samples were
pooled and intravenously (IV) transplanted into sublethally irradiated
NOD/SCID mice using 9 cell concentrations ranging from 1,660 to 225,000 CD34+ UCB cells per mouse. Per cell dose, the median number
of mice used was 6 (2 to 34). After 35 days, the femora were isolated and BM cells harvested. The percentage of multilineage engraftment was
determined using immunophenotypic analysis for each individual mouse.
Mice containing 1% or more CD45+ cells were considered
positive for human engraftment. During the engraftment period, ie, 35 days posttransplant, the mice were not supplemented with cytokines. The
frequency of the SRC in UCB in our laboratory was determined by using
Poisson statistics. Part of the repopulation data used has been
published by Verstegen et al.9
Assessment of the seeding efficiency of the transplanted cells.
For determining the seeding efficiency in NOD/SCID mice of all 5 grafts studied, we determined the number of CAFC subsets and CFC per
graft. Simultaneously, we irradiated 2 recipient NOD/SCID mice per
sample with 9 Gy by a 137Cs source (Gammacell; Atomic
Energy of Canada, Ottawa, Canada). This dose was chosen to maximally
reduce the recipient hematopoietic activity, allowing the detection of
low numbers of human seeded stem cells. Two to 5 hours after
irradiation, the mice were transplanted. In the experiments assessing
the seeding of CD34+ UCB cells as determined by flow
cytometry, the mice were either 3.5 or 9 Gy irradiated. The number of
transplanted cells per mouse varied in case of unsorted UCB between 63 and 111 × 106 nc, for BM between 30 and 48 × 106 nc, for PBSC between 30 and 94 × 106
nc, and for NOD/SCID BM between 46 and 63 × 106 nc.
The median percentage of CD34+ cells in the unsorted UCB
graft was 1.7, in BM 5.3, and in PBSC 1.5. For the CD34+
grafts, the cell numbers varied between 375,000 and 800,000 pure CD34+ nc per mouse. In case of transplanting unsorted UCB,
BM, PBSC, NOD/SCID BM, and CD34+ selected UCB nc 3, 5, 6, 4, and 7 individual samples have been used, respectively. At 22 to 24 hours after transplantation,10,11 the mice were killed and
femora and spleen were isolated. The BM cells were harvested by
crunching the bones as described above. The spleen was carefully cut in
parts and sieved through a 100-µm mesh filter. The cells were then
washed and resuspended in CAFC medium or PBS containing 0.5% bovine
serum albumin (BSA) and 2% normal human serum (NHS; obtained from
healthy volunteers) in case of preparing the cells for
immunophenotyping. In the CAFC assay, 1/10 femur or 1/50 spleen were
plated in the first dilution. For the CFC assay, 1/10 and 1/100 femur
and 1/50 and 1/500 spleen were plated. The irradiation control was
negative in 6 of 6 experiments. For determining the seeding efficiency
to the total BM, we assumed that 1 femur contains approximately 6% of
the total BM cellularity.12 The BM and spleen seeding
efficiencies were calculated on the basis of the number of infused and
retrieved CAFC and CFC. When CD34+ UCB cells were infused
and the phenotypic seeding efficiency was determined, the number of
retrieved CD34+ UCB cells (as determined using Flow Count
Fluorospheres; Coulter Immunotech, Miami, FL) was divided by the number
of CD34+ UCB cells infused corrected for the purity of the
population. Seeding efficiency is expressed as percentage.
Immunofluorescence analysis.
Cells were stained with anti-CD34-fluorescein isothiocyanate (FITC) or
phycoerythrin (PE), anti-CD45-FITC (Coulter Immunotech, Mijdrecht, The
Netherlands) or anti-CD38-PE (Becton Dickinson, San Jose, CA) or FITC
(Coulter Immunotech) by incubating 105 to 106
nc for 30 minutes on ice. The incubations were performed in PBS containing 0.5% BSA (Sigma) and 2% NHS. After incubation, the cells
were washed once in PBS and 0.5% BSA and resuspended in 0.3 mL PBS.
Analysis was performed using a Facscan (Becton Dickinson). A total of
10 to 25,000 events was acquired per sample. In case of using flow
cytometry for determining the seeding efficiency of CD34+
selected UCB cells in the BM of the NOD/SCID mouse, a minimum of
750,000 events was acquired. Seven-aminoactinomycin D (7-AAD; Molecular
Probes, Eugene, OR) was used to exclude the dead cells.
Hematopoietic growth factors.
Purified recombinant human granulocyte-macrophage colony-stimulating
factor (GM-CSF) and murine stem cell factor (SCF) were kindly provided
by Genetics Institute (Cambridge, MA). Human granulocyte colony-stimulating factor (G-CSF) and human interleukin-3 (IL-3) were
gifts from Amgen (Thousand Oaks, CA) and Gist Brocades (Delft, The
Netherlands), respectively.
Human CFC assay.
Quantification of the number of colony-forming units-granulocyte
macrophage (CFU-GM) and burst-forming units-erythroid (BFU-E) was
performed using a semisolid (1.2% methylcellulose; Methocel, Stade,
Germany) culture medium (Iscove's modified Dulbecco's medium [IMDM]; GIBCO) at 37°C and 5% CO2. The cultures
contained 30% FCS supplemented with penicillin (100 U/mL; GIBCO),
streptomycin (100 µg/mL; GIBCO),  -mercapto-ethanol (me; 5 × 10 5 mol/L; Merck, Darmstadt, Germany),
erythropoietin (1 U/mL; Boehringer, Mannheim, Germany), IL-3 (15 ng/mL), G-CSF (50 ng/mL), GM-CSF (5 ng/mL), and murine SCF (100 ng/mL)
all at final concentrations. CFU-GM and BFU-E consisting of more than
50 cells were counted on day 14 of culture in the same dish.
Murine CFC assay.
The number of CFC present in the murine BM and spleen cells harvested
was determined by plating the cells in semisolid cultures consisting of
1.2% (wt/vol) methylcellulose (Methocel) in IMDM. The medium was
supplemented with 10% (vol/vol) pokeweed mitogen-stimulated mouse
spleen-conditioned medium and 20% horse serum (HS; GIBCO). The
cultures were kept at 37°C and 10% CO2. Colonies
consisting of 50 cells and more were counted at day 7 and day 14 of culture.
Stromal feeders.
The Flask Bone Marrow Dexter (FBMD-1) murine stromal cell
line was used as described earlier.2 In short, stromal
feeders were prepared by seeding 103 FBMD-1 cells per well
into flat-bottom 96-well plates (Falcon, Lincoln Park, NJ) from
log-phase cultures. Culture plastics destined for establishment of
FBMD-1 stromal feeders were incubated overnight at 4°C with 0.3%
gelatin in demineralized water to improve adherence of the stromal
layer. The FBMD-1 cells were cultured in FBMD-1 medium consisting
of IMDM with glutamax-1 (GIBCO) supplemented with penicillin (100 U/mL), streptomycin (100 µg/mL), me (104
mol/L), 10% FCS, 5% HS (Integro, Zaandam, The Netherlands), and hydrocortisone 21-hemisuccinate (105 mol/L; Sigma).
After 7 to 10 days of culture at 33°C and 10% CO2, the
stromal layers had reached confluence and were used for the CAFC assay.
CAFC assay.
Confluent stromal layers of FBMD-1 cells in flat-bottom 96-well plates
were overlaid with NOD/SCID BM nc, UCB nc (unsorted or
CD34+ selected), BM nc, or PBSC in a limiting dilution
setup. For the primary CAFC assays of the NOD/SCID BM nc, UCB nc, BM
nc, and PBSC, the input values ranged from 27,000 to 50,000 nc per well and 250 cells per well in case CD34+ UCB cells. The portion
of the harvested BM and spleen of the transplanted NOD/SCID mice used
in the first dilution of the CAFC assay is indicated above. Twelve
dilutions, 2-fold apart were used for each sample with 15 replicate
wells per dilution. The cells were cultured at 33°C and 10%
CO2 for 5 weeks in case of mouse cells and 6 weeks for the
human CAFC with weekly half-medium changes. For the murine CAFC assay,
FBMD-1 medium was used and in case of performing the human CAFC assay,
the medium consisted of FBMD-1 medium supplemented with IL-3 and G-CSF
at final concentrations of 10 ng/mL and 20 ng/mL, respectively. The
percentage of wells with at least 1 phase-dark hematopoietic clone of
at least 5 cells (ie, cobblestone area) beneath the stromal layer was
determined weekly for the mouse CAFC and every 2 weeks for the human
CAFC. Frequencies of the CAFC subsets were calculated using Poisson statistics as described previously.1
Data analysis.
Microsoft Excel 97 (Microsoft, Redmond, WA) and SPSS for Windows
Release 7.5.2. (SPSS Inc, Chicago, IL) were used for data analysis. Data are expressed as median (range).
Statistical comparisons were performed according to Mann Whitney
U-test. The 2-sided P value was determined testing the null
hypothesis that the 2 population medians are equal. P
values <.05 were considered significant.
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RESULTS |
SRC frequency in UCB.
The SRC frequency as determined by limiting dilution analysis in our
laboratory is approximately 1 per 6.6 × 106 unsorted
UCB nc. The threshold for human engraftment used to distinguish
positive from negative mice is 1% CD45+ nc in the BM of
the NOD/SCID mouse as assessed by flow cytometry.
Low seeding efficiency of NOD/SCID BM cells in NOD/SCID mouse BM and
spleen.
To determine the seeding efficiency of syngeneic hematopoietic cells in
the NOD/SCID mouse strain, NOD/SCID BM nc were isolated from 6- to
9-week-old NOD/SCID mice and processed according to the seeding
efficiency protocol as described in Materials and Methods. The seeding
efficiency to the total BM of the NOD/SCID mouse tended to decrease
with increasing immaturity of the progenitor cell subset studied
(Fig 1). The median (range) seeding
efficiency to the total BM for CFC day 7 (d 7) and d 14 was 19.5 (17.6 to 24.1) and 22.8 (13.1 to 28.4), respectively. In contrast, the seeding efficiency of CAFC wk 5 was 8.7 (2.0 to 9.2). For the seeding
efficiency of the progenitor subsets to the murine spleen, we did not
observe significant differences. The seeding efficiency of d 7 CFC and
d 14 CFC to the spleen was 1.4 (0.5 to 2.4) and 1.2 (0.9 to 2.5),
respectively. The percentage of CAFC wk 5 lodging to the spleen was 2.9 (0.7 to 4.0).
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| Fig 1.
Seeding efficiency of unsorted NOD/SCID BM cells in total
bone marrow ( ) and spleen ( ) of NOD/SCID mice. The data represent
the median and range of 4 separate experiments.
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Seeding efficiency of unsorted human progenitors in total BM and
spleen of NOD/SCID mice.
All of the progenitor subsets analyzed from unsorted UCB nc showed a
lower seeding efficiency to the BM and the spleen than was observed in
the NOD/SCID syngeneic setting (Fig 2). The
seeding efficiency of d 14 CFC and CAFC wk 6 to the total BM of the
NOD/SCID mouse was in case of unsorted UCB nc 3.8 (3.8 to 5.5) and 4.4 (3.5 to 6.3), in BM nc 0.9 (0.1 to 1.4) and 0.8 (0.3 to 1.7), and in
PBSC 2.1 (0.1 to 3.9) and 5.3 (1.4 to 13.6), respectively. The d 14 CFC
and CAFC wk 6 seeding efficiency to the spleen was in case of unsorted
UCB nc 0.7 (0.6 to 1.0) and 0.5 (0.4 to 0.8), for BM nc 0.3 (0.03 to
0.9) and 0.2 (0.1 to 0.2), and for PBSC 0.6 (0.1 to 1.2) and 0.5 (0.1 to 1.8). Thus, of all infused progenitors derived from, for example,
unsorted UCB nc, only around 4 of every 100 CAFC infused will home to
the total BM and only 5 in every 1,000 will lodge to the spleen of the
NOD/SCID mouse. The seeding efficiency of the various hematopoietic
progenitor cell types from PBSC was comparable to the seeding
efficiency of UCB nc, both to total BM and spleen (Fig 2). However,
PBSC grafts showed larger variability in seeding to BM and spleen as
compared with UCB grafts. As with UCB cells, no significant differences
could be observed among the subsets assayed.
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| Fig 2.
Seeding efficiency of human unsorted UCB nc, PBSC, and BM
nc in total NOD/SCID BM () and spleen (). The data are depicted
as median and range. n, number of separate experiments.
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Also the seeding efficiency of human BM progenitors to the murine BM
and the spleen showed no significant differences in seeding efficiency
among the various stem cell subsets studied.
Seeding efficiency of CFC and CAFC subsets in CD34+
selected UCB cells.
As it may be argued that the seeding efficiency may change in relation
to the cell number infused, we have compared the seeding efficiency of
CFC and CAFC subsets from CD34+ selected and unsorted UCB
cells (Fig 3). No significant difference could be observed. As observed with the unsorted grafts, the different progenitor subsets contained in the CD34+ grafts home in
comparable percentages to the BM of the NOD/SCID mouse.
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| Fig 3.
Comparison of the seeding efficiency of human
CD34+ selected ( ) and unsorted ( ) UCB cells in
total NOD/SCID BM. The data are depicted as median and range. Three
separate experiments for either population were performed.
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The effect of 3.5 Gy versus 9 Gy irradiation conditioning on the
seeding efficiency of CD34+ UCB cells as determined using
flow cytometric analysis.
Determination of the repopulating potential of human cells in the
NOD/SCID mouse model commonly includes conditioning using a sublethal
total body irradiation of 3.5 Gy. In our experiments to determine the
seeding efficiency of human grafts in the NOD/SCID mouse, we irradiated
the mice with a supralethal irradiation of 9 Gy to fully eradicate any
measurable constitution by murine progenitors. This dose of irradiation
would then assure detection of human progenitor cells exclusively, as
we are not able to distinguish murine and human progenitors using the
CFC and CAFC assay as used here. To assess whether the level of
irradiation affected the seeding efficiency of human progenitors in the
NOD/SCID mice, we compared the seeding efficiency of CD34+
selected UCB cells as assessed by flow cytometry in 3.5 Gy and 9 Gy
irradiated NOD/SCID mice. Figure 4 shows
the flow cytometric analysis used to determine the number of
CD34+ UCB cells retrieved from the NOD/SCID mouse BM 24 hours after transplantation. In dot plot A, living, ie, 7-AAD negative,
CD34-PE positive cells are gated and shown in dot plot B. The
CD45dim cells possessing large forward light scatter
properties were gated to prevent false positive events due to the high
autofluorescence of murine BM cells and shown in a CD45-FITC versus
CD34-PE dot plot (C). Dot plot C shows a clear double positive
population representing the CD34+ UCB cells that had homed
to the NOD/SCID mouse BM.
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| Fig 4.
Flow cytometric analysis used to determine the number of
CD34+ UCB cells retrieved from the NOD/SCID mouse BM 24 hours after transplantation. Dead cells were excluded using 7-AAD. In
dot plot A, living CD34-PE positive cells are gated and shown in dot
plot B. CD45dim cells possessing large forward light
scatter properties are gated and shown in a CD45-FITC versus CD34-PE
dot plot (C). Dot plot C clearly shows that all of the gated cells in
plot B belong to a clear double positive population representing the
CD34+ UCB cells retrieved from the NOD/SCID mouse BM.
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Figure 5 shows that the median seeding
efficiency of CD34+ UCB cells in 3.5 Gy irradiated
recipients (2.3%) as compared with 9 Gy conditioned NOD/SCID (2.5%)
mice as measured by flow cytometry was not significantly different.
Furthermore, the seeding efficiency of CD34+ selected UCB
cells as determined by the CFC and the CAFC assay is similar to that
assessed by flow cytometric analysis except for the CAFC wk 6 (P = .04). However, in CD34+ selected UCB cells,
the CAFC wk 2 frequency relates to the CAFC wk 6 frequency as 4:1.
Therefore, the seeding efficiency of CD34+ UCB cells as
determined using flow cytometry will be in the range of the CAFC wk 2 seeding.
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| Fig 5.
Seeding efficiency of human CD34+ UCB cells
in total BM of NOD/SCID mice: comparison of flow cytometric analysis
with CFC and CAFC data. The data are depicted as median and range of 3 separate experiments for the CFC and CAFC assays; 6 experiments for the
3.5-Gy irradiated NOD/SCID mice, and 7 experiments for the 9-Gy
irradiated NOD/SCID mice. *, Significantly different from CAFC wk 6.
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DISCUSSION |
We have shown that the seeding efficiency of human progenitor cells in
the BM and spleen of NOD/SCID mice is extremely low. As the different
progenitor cell subsets had comparable low seeding rates, it is
conceivable by extrapolation that only a few of all infused NOD/SCID
repopulating cells will home to the marrow of these mice. Thus, the
assumption that every SRC infused will indeed contribute to
repopulation should be critically met.5 Rather, our data
indicate that the published frequency estimates of human SRC in UCB,
BM, and PBSC represent a dramatic underestimation. If these frequency
estimates were corrected by the respective seeding efficiencies as
currently published, they would be 18-fold to 125-fold higher than
presently accepted.
We have used 1% CD45+ as a threshold for human engraftment
in BM of the NOD/SCID mouse to distinguish positive from negative mice.
This threshold is 10-fold and 20-fold higher than the threshold used by
Conneally et al6 and Wang et al,5 respectively. The lower detection threshold used by Wang et al and Conneally et al
may explain why these investigators arrived at a higher SRC frequency estimate.
The seeding efficiency of NOD/SCID BM nc to the BM and the spleen of a
syngeneic recipient is lower than we have observed in other murine
syngeneic transplantation settings.7 Particularly, the
seeding efficiency to the spleen is strikingly low. The reason for this
discrepancy is as yet unresolved; however, there are several possible
explanations for this observation. (1) There could be intrinsic
differences of the primitive stem cells between the 2 mouse strains
influencing their homing behavior. (2) The data could indicate that the
microenvironment in the NOD/SCID mouse may be less conducive for
allowing homing of infused stem cells.
We observed similar seeding efficiencies to the NOD/SCID BM of CFC and
CAFC subsets, irrespective of whether they were contained in
CD34+ selected or unsorted UCB cells. These data suggest
that CD34 UCB cells in the unsorted grafts did not
affect the homing of the progenitor subsets contained in the
CD34+ population.
By using flow cytometry, we were able to compare the seeding efficiency
of CD34+ selected UCB cells in 3.5 Gy and 9 Gy irradiated
NOD/SCID mice. Furthermore, the seeding efficiency of CD34+
selected UCB cells obtained with immunophenotypic analysis was not
significantly different from the data obtained with the CFC and CAFC
assays (except for CAFC wk 6). Although the data are highly suggestive,
they do not exclude the possibility that CD34+ UCB cells
show differences in cloning efficiency in the CFC and CAFC assay after
seeding in BM of a 3.5-Gy irradiated mouse as compared with seeding in
BM of a 9-Gy irradiated recipient.
Homing of stem and progenitor cells to the BM after IV transplantation
has been defined as the cells' ability to seek marrow stroma
selectively, to subsequently lodge within it, and initiate hematopoiesis.13 As investigated in the murine system, this process is believed to include 2 phases. The first phase consists of
the recognition of the endothelial cells by the hematopoietic cells
possibly through a lectin receptor with galactosyl
specificity.14,15 The second phase is the interaction of
galactosyl/mannosyl-specific homing receptor to the extracellular
matrix of the BM16 and the interaction of the very late
antigen-4 (VLA-4) receptor with vascular-cell-cell adhesion molecule
(VCAM).17-19 Therefore, the process of homing seems to be
highly specific. In other organs (ie, liver, lung, and kidney),
hematopoietic progenitor cells can be temporarily detected after their
IV transplantation and disappear within 48 hours after
transplantation.20 Their sequestration to the latter organs
may be determined by other mechanisms, including binding to galactosyl
receptors.21
Also the homing process to the spleen seems to be regulated differently
than in the marrow. Recognition of galactosyl/mannosyl residues16 or the fibronectin receptor
(VLA-4)20 seems not to be involved. The special anatomical
structure of the spleen is suggested to be one of the determinants of
spleen cell homing.
In our experiments, we have injected human cells into a sublethally and
supralethally irradiated NOD/SCID mouse. Several publications indicate
that the molecules involved in the homing process are highly conserved
in evolution.22-24 Therefore, it is likely that the
transplanted human cells will home to the BM and spleen of the NOD/SCID
mouse in a specific manner.
Our data indicate otherwise. As the total BM of a mouse may be
estimated to weigh around 350 mg, while an NOD/SCID mouse 9 weeks old
weighs around 18 g, the total BM is around 2% of the total weight of
the animal. The weight of the spleen is around 1% of the animal
weight. If the homing of the human cells is random, then the seeding
efficiency to the total BM will be 2% and to the spleen 1%. However,
when we take into account that tissues like fat, skin, and brain of the
mouse are probably less accessible for progenitor cells, while
additionally the capillary beds of the BM and the spleen may be rather
extensive, we will expect a slightly higher seeding efficiency than
these figures if seeding is a nonspecific process. In agreement with
the calculated seeding percentages, the median seeding efficiencies of
the stem cells in the human grafts studied to the total BM vary between
1% and 7% and to the spleen between 0.2% and 0.9%. These data
suggest that human progenitors do not home preferentially to the total BM and spleen of the NOD/SCID mouse, but that seeding of human hematopoietic progenitors may predominantly occur on the basis of
organ-weight and capillary bed complexity.
The expression of adhesion molecules is different among
CD34+ cells derived from UCB and BM as compared with PBSC.
PBSC shows lower expression of VLA-4, leukocyte function anigen-1
(LFA-1), intercellular adhesion molecule-1 (ICAM-1), and LFA-3 than
BM.25,26 The adhesion molecule expression pattern in UCB nc
shows similarities with BM nc, although UCB-derived
CD34+/CD38 cells express higher levels
of L-selectin (CD62L) and LFA-1 (CD11a) than their BM
counterparts.27 This would mean that if the homing process
of the human cells in the NOD/SCID mouse is specific, we might expect
comparable seeding efficiencies of UCB and BM nc and a lower seeding of
PBSC. In contrast, the seeding efficiencies of UCB nc and PBSC are
comparable, while the seeding of BM nc in the BM of the NOD/SCID mouse
is very low.
It has been published that expression of the molecules that recognize
galactosyl and mannosyl residues changes during
differentiation.28 This could cause differences in seeding
efficiencies among progenitor and stem cell subsets. However, the fact
that we did not observe this provides us with another strong indication
that homing of the human cells in the BM and the spleen of the NOD/SCID
mouse is not specific.
Our data suggest that the postulated low frequencies of human long-term
repopulating stem cells may largely result from their low seeding
efficiency in the BM of NOD/SCID mice. In addition, it should be
realized that the proposed SRC frequency in the various human grafts is
the resultant of (1) seeding efficiency, (2) repopulating ability of
the transplanted cells, (3) exogenous factors facilitating engraftment,
(4) detection methods, (5) detection thresholds, and (6) definition of
a SRC. In light of this notion, the estimated human stem cell frequency
could even be higher than the SRC frequency corrected for the seeding
efficiency of that particular graft.
It has been recently suggested that the SRC may represent a different
and more primitive stem cell subset than are assessed by the LTC-IC and
CAFC assay on the basis of a variety of observations.5 Thus, SRC would be more difficult to transduce and occur at far lower
frequencies than the LTC-IC or CAFC wk 6. Our recent data on comparable
gene expression in CAFC wk 6 and SRC,29 together with our
present data, would suggest that the SRC and CAFC wk 6 may differ far
less than previously claimed and thus may represent overlapping stem
cell populations.
| |
ACKNOWLEDGMENT |
The authors thank Dr A.Th. Alberda and the staff of the St Franciscus
Hospital (Rotterdam, The Netherlands) for collection of cord blood
samples used in this study. We also acknowledge Els van Bodegom for
taking care of the NOD/SCID mice. Drs M.M.A. Verstegen and N. Kusadasi
are gratefully acknowledged for sharing their data for SRC frequency analysis.
| |
FOOTNOTES |
Submitted February 16, 1999; accepted June 28, 1999.
Supported by Grant No. 95-1020 from the Dutch Cancer Society.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Rob E. Ploemacher, PhD, Institute of
Hematology, Room Ee 1391, Erasmus University Rotterdam, Dr.
Molenwaterplein 50, PO Box 1738, 3000 DR Rotterdam, The Netherlands;
e-mail: ploemacher{at}hema.fgg.eur.nl.
| |
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