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Blood, Vol. 93 No. 11 (June 1), 1999:
pp. 3736-3749
Engraftment in Nonobese Diabetic Severe Combined Immunodeficient
Mice of Human CD34+ Cord Blood Cells After Ex Vivo
Expansion: Evidence for the Amplification and Self-Renewal of
Repopulating Stem Cells
By
Wanda Piacibello,
Fiorella Sanavio,
Antonella Severino,
Alessandra Danè,
Loretta Gammaitoni,
Franca Fagioli,
Eliana Perissinotto,
Giuliana Cavalloni,
Orit Kollet,
Tsvee Lapidot, and
Massimo Aglietta
From the Department of Biomedical Sciences and Human Oncology,
University of Torino Medical School, Torino; the Hematology/Oncology
Section, Mauriziano Hospital, Torino; the Pediatric Department; the
Institute for Cancer Research and Treatment (IRCC), Candiolo, Torino,
Italy; and the Department of Immunology, the Weizmann Institute of
Science, Rehovot, Israel.
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ABSTRACT |
Understanding the repopulating characteristics of human
hematopoietic stem/progenitor cells is crucial for predicting their performance after transplant into patients receiving high-dose radiochemotherapy. We have previously reported that CD34+
cord blood (CB) cells can be expanded in vitro for several months in
serum containing culture conditions. The use of combinations of
recombinant early acting growth factors and the absence of stroma was
essential in determining this phenomenon. However, the effect of these
manipulations on in vivo repopulating hematopoietic cells is not known.
Recently, a new approach has been developed to establish an in vivo
model for human primitive hematopoietic precursors by transplanting
human hematopoietic cells into sublethally irradiated nonobese diabetic
severe combined immunodeficient (NOD/SCID) mice. We have examined here
the expansion of cells, CD34+ and
CD34+38 subpopulations, colony-forming
cells (CFC), long-term culture initiating cells (LTC-IC) and the
maintenance or the expansion of SCID-repopulating cells (SRC) during
stroma-free suspension cultures of human CD34+ CB cells
for up to 12 weeks. Groups of sublethally irradiated NOD/SCID mice were
injected with either 35,000, 20,000, and 10,000 unmanipulated
CD34+ CB cells, which were cryopreserved at the start of
cultures, or the cryopreserved cells expanded from 35,000, 20,000, or
10,000 CD34+ cells for 4, 8, and 12 weeks in the presence
of a combination of early acting recombinant growth factors (flt 3/flk2
ligand [FL] + megakaryocyte growth and development factor [MGDF] ± stem cell factor [SCF] ± interleukin-6 [IL-6]).
Mice that had been injected with  20,000 fresh or cryopreserved
uncultured CD34+ cells did not show any sign or showed
little engraftment in a limited number of animals. Conversely, cells
that had been generated by the same number of initial
CD34+ CB cells in 4 to 10 weeks of expansion cultures
engrafted the vast majority of NOD/SCID mice. The level of engraftment,
well above that usually observed when the same numbers of uncultured cells were injected in the same recipients (even in the presence of
irradiated CD34 cells) suggested that primitive
hematopoietic cells were maintained for up to 10 weeks of cultures. In
addition, dilution experiments suggest that SRC are expanded more than
70-fold after 9 to 10 weeks of expansion. These results support and
extend our previous findings that CD34+ CB stem cells
(identified as LTC-IC) could indeed be grown and expanded in vitro for
an extremely long period of time. Such information may be essential to
design efficient stem cell expansion procedures for clinical use.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
AN ESSENTIAL PROPERTY of hematopoietic
stem cells is their ability to divide without significant alteration of
their proliferative potential or differentiation state.
Characterization and quantitation of these progenitor cells is
fundamental to our understanding of the developmental sequence, and it
is of great importance for human hematopoietic cell transplantation, ex
vivo expansion, and gene therapy.1-4 To date, quantitative
analysis of human primitive hematopoietic cells has been limited to in vitro studies using colony assays (colony-forming cells [CFC]) or
long-term cultures (LTC). CFC assays detect only committed and
multipotent progenitors. LTC assays detect more primitive cells
(LTC-initiating cells [IC]), capable of generating myeloid colonies
for at least 5 weeks of culture on competent feeder
layers.5,6
The transplantation assay available in the mouse system has been
instrumental in defining and characterizing the most primitive elements
of the hematopoietic system.7 Recently, a similar in vivo
approach, derived from the work in the mouse, has become available for
humans. As a result, several groups have transplanted human
hematopoietic precursors into different mouse mutants in an attempt to
develop a reproducible transplantation assay.7-9 In
particular, the intravenous injection of human hematopoietic precursors
in sublethally irradiated severe combined immunodeficient (SCID) and
nonobese diabetic/SCID (NOD/SCID) mice has resulted in the engraftment
of primitive human cells that proliferate and differentiate to multiple
lineages in the murine bone marrow (BM) and spleen.10-13
The transplanted human cells home to and engraft the murine BM, where
they proliferate and differentiate to produce large numbers of LTC-IC,
CFC, immature and mature myeloid, erythroid, and lymphoid cells without
the influence of exogenously supplied human growth
factors.14 The engrafted human cells have been defined
SCID-repopulating cells (SRC)15 or, in a similar
quantitative assay, competitive repopulating units (CRU).16
Although the SRC (or CRU) represents a very primitive hematopoietic
cell, the exact place in the stem cell hierarchy and its relation to
early in vitro hematopoietic progenitors, such as LTC-IC, is not fully understood. SRC have been reported to be biologically distinct from and
more primitive than most CFC and LTC-IC: SRC are exclusively CD34+CD38 in contrast to CFC and LTC-IC,
which are found also in the CD34+CD38+
fraction.17-19 By contrast, according to Eaves's data,
even though the cells identified by the LTC-IC and the CRU
assay may not necessarily represent identical cell
populations, they belong to both CD34+CD38+
and CD34+CD38 subpopulations and seem to
increase in response to the same culture conditions in
vitro.16
We conclude that this experimental transplantation assay
that measures the repopulating potential of various human progenitor fractions is most valuable in increasing our understanding of several
biological properties of hematopoietic progenitors for both
experimental and clinical hematology.
A second very important issue, to date, is the identification of
culture conditions that support the self-renewal and the expansion of
human hematopoietic stem cells. In particular, cord blood (CB) has
recently attracted attention as a source of hematopoietic stem cells
for both transplantation and gene therapy
applications.20-24 However, concern that a single CB
collection may not be sufficient to guarantee engraftment of adult
allogeneic recipients has also stimulated considerable interest in
expanding CB stem cell number in vitro. Experiments conducted in our
laboratories as well as data from others have recently shown that
CD34+ (or CD34+ CD38) CB
cells can indeed be expanded in fairly well-defined culture conditions.25-29 The common denominator of the expansion
systems was the absence of stroma layers and hence the use of
combinations of recombinant early acting hematopoietic growth factors.
A net expansion of LTC-IC was observed in all cases.25-29
In addition, it was shown that 4-day or 5- to 8-day stroma-free liquid
cultures of CB cells in the presence of flt3/flk2 ligand (FL), stem
cell factor (SCF), interleukin-3 (IL-3), IL-6 without29 or
with granulocyte colony-stimulating factor (G-CSF)16
supported LTC-IC expansion and also a modest, albeit significant
expansion, of CRU in NOD/SCID mouse recipients. Conversely, it has been
reported that cocultures of human BM and CB cells with allogeneic human
stroma resulted in a reduced repopulating capacity of cocultured cells,
which, by contrast, contained an equal or even higher number of CFC and LTC-IC as compared with uncultured cells.30 On the other
hand, very recently Xu et al31 reported that in vivo
long-term repopulating hematopoietic stem cells from CB could be
maintained for at least 4 weeks when cocultured on a stroma cell line
derived from the aorta-gonad-mesonephros region of mouse embryo.
The aim of our studies was to investigate whether the combinations of
early acting growth factors, which in our previous studies proved
capable of inducing a massive and prolonged expansion of hematopoietic
progenitors and more primitive LTC-IC from CB cells,27,28 could also maintain the in vivo repopulating ability of human stem
cells, or even amplify their number.
We show that CD34+ CB cells can be expanded for up to 10 weeks in stroma-free cultures in the presence of FL, megakaryocyte growth and development factor (MGDF), SCF, and IL-6
without losing their in vivo repopulating potential. Furthermore, our
studies show that considerable expansion of SRC is obtainable in vitro, as sublethally irradiated NOD/ SCID mice are consistently and reproducibly repopulated by the equivalent of 1,250, 625, and 312 initial CD34+ CB cells, which have been expanded in vitro
for up to 9 to 10 weeks.
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MATERIALS AND METHODS |
Human cells.
Human BM was obtained by aspiration from the posterior iliac crest of
fully informed hematologically normal donors. Umbilical CB was obtained
at the end of full-term deliveries, after clamping and cutting of the
cord, by drainage of blood into sterile collection tubes containing the
anticoagulant citrate-phosphate dextrose.
CD34+ cell purification.
Mononuclear cells (MNC) were isolated from CB using Ficoll Hypaque
(density, 1077; Nyegaard, Oslo, Norway) density centrifugation. Cells
were subjected to two cycles of plastic adherence (60 minutes each);
they were then washed with Hanks' Balanced Salt Solution (HBSS, GIBCO
BRL, Grand Island, NY). The CD34+ MNC fraction was isolated
with superparamagnetic microbead selection using high-gradient magnetic
field and miniMACS column (Miltenyi Biotech, Gladbach, Germany). The
efficiency of the purification was verified by flow cytometry
counterstaining with a CD34-phycoerythrin (PE; HPCA-2;
Becton Dickinson, San Jose, CA) antibody. In the cell fraction
containing purified cells, the percentage of CD34+ cells
ranged from 90% to 98%.
Recombinant human cytokines.
The following recombinant purified human cytokines were used in these
studies: recombinant human (rh) stem cell factor (rhSCF) and rh
megakaryocyte growth and development factor (MGDF) were a generous gift
from Amgen (Thousand Oaks, CA); recombinant human granulocyte
colony-stimulating factor (rhG-CSF) was from Genzyme (Cambridge, MA);
recombinant human granulocyte-macrophage colony-stimulating factor
(rhGM-CSF), recombinant human interleukin-6 (rhIL-6) and recombinant
human interleukin-3 (rhIL-3) were from Sandoz (Basel, Switzerland);
recombinant human erythropoietin (rhEPO; EPREX) was from Cilag (Milan,
Italy); recombinant human FLT3-ligand (rhFL) was kindly provided by
S.D. Lyman (Immunex Corp, Seattle, WA).
Animals.
NOD/LtSz scid/scid (NOD/SCID) mice were obtained from
Charles River Italia (Calco, Italy) and maintained in the animal
facilities at Antoine Marxer-RBM (Colleretto Giacosa, Italy). In part
of the studies, mice were bred and maintained at the animal facilities of the Weizman Institute of Science (Rehovot, Israel).
All animals were handled under sterile conditions and maintained in
cage microisolators. Mice to be transplanted were irradiated at 6 to 8 weeks of age with 350 to 375 cGy of total body irradiation from a
137Cs source and then within 24 hours were given a single
intravenous injection of: (1) human CD34+ CB cells, which
had been previously separated from several CB samples, then pooled, and
cryopreserved (control cells). After thawing, 97% to 99%
CD34+ cells were viable by trypan blue dye exclusion; (2)
cells were harvested from expansion cultures as described. Also the
latter cells were cryopreserved and injected at the same time as the control cells. When low numbers of unmanipulated CD34+
cells were to be transplanted, at least 2 × 105
irradiated CD34 cells were coinjected as carrier cells.
Mice were killed 6 to 8 weeks posttransplant for assessment of the
number and types of human cells detectable in both femurs, tibias, and
spleen. As a control for the frequency of SRC in cryopreserved CD34+ CB cells, the same numbers of fresh CD34+
cells from pooled CB samples were injected in similarly pretreated NOD/SCID recipients; the frequency of SRC was not significantly different from that found in cryopreserved CD34+ cells.
Flow cytometric detection of human cells in murine tissues.
BM cells were flushed from the femurs and tibias of each mouse to be
assessed using a syringe and a 26-gauge needle. To prepare cells for
flow cytometry, contaminating red blood cells were lysed with 8.3%
ammonium chloride and the remaining cells were then washed in HBSS with
0.1% bovine serum albumin (BSA; Sigma Chemical Co, Milan, Italy),
0.01% sodium azide (HFN). The cells were then resuspended at 1 to 2 × 106 cells/mL and incubated with mouse IgG (Fluka
Chemika Biochemika, Buchs, Switzerland) and with 5% human serum (HS),
to block nonspecific binding to Fc receptor. Cells were then incubated
with monoclonal antibodies (MoAb) specific for human CD45, CD71, and
glycophorin-A (GpA), directly labeled with fluorescein isothiocyanate
(FITC) or PE (all from Dako A/S, Glostrup, Denmark) for 30 minutes at 4°C to assess the total population of human
hematopoietic cells. Cells stained with an anti-CD45 conjugated to an
R-phycoerythrin-Cy5 tandem conjugate were simultaneously stained with
an anti-human-CD34-PE (Becton Dickinson) and CD19-PE (Dako) for
quantitation of the total human CD34+ and CD19+
cell populations. In some mice, additional aliquots were stained with
anti-human-CD33-PE (Dako), CD41-FITC in combination with anti-human-CD45-Cy5 RPE to allow discrimination of subpopulations within the CD45 gate and with CD71-FITC plus  -GpA-PE.30
Some cells from each suspension were similarly incubated with
irrelevant (control) MoAbs labeled with FITC and PE. Cells from an
unmanipulated NOD/SCID mouse were also stained with each of the MoAbs
used for detecting positively stained human cells. Only levels of
fluorescence which excluded 99.9% of all of these negative controls
were considered as specific. After staining, all cells were washed once
in sodium azide (HFN) containing 2 µg/mL propidium iodide (PI) to
allow dead (PI+) cells to be excluded from analyses. Flow
cytometric analysis was performed using a FACScan cytometer (Becton
Dickinson). At least 5,000 events were acquired for each analysis. When
fluorescent cells represented only a minority of the total population
( 0.1%) many more events (at least 20,000) were analyzed.
Hematopoietic cell cultures.
Assays for granulopoietic, erythroid, megakaryocytic, and multilineage
(granulocyte-erythroid-macrophage-megakaryocyte) colony-forming units
(CFU-GM, BFU-E, CFU-Mk, and CFU-GEMM, respectively) were usually
performed as follows. For CFU-GM, 1 × 103
CD34+ CB cells of the initial cell suspension or suitable
aliquots of the stroma-free long-term cultures were cultured at 4 plates per point in 3% agar, 15% fetal calf serum (FCS) (HyClone,
Logan, UT) in Iscove's Modified Dulbecco Medium (IMDM). For CFU-Mk,
the same number of cells was cultured in plasma-clot assay (four dishes per point) as previously described.27 For BFU-E and
CFU-GEMM, the same number of cells was cultured in 1.3%
methylcellulose (Fluka) and IMDM containing 30% FCS at 37°C in a
humidified atmosphere at 5% CO2 in air.27
Colony scoring was performed on day 12 for CFU-Mk (at the
immunofluorescent microscope after staining with an FITC-conjugated
MoAb recognizing human GPIIbIIIa) and on day 14 for CFU-GM, BFU-E, and
CFU-GEMM.27,28 Several growth factors were added at optimum
concentrations to sustain the formation of BFU-E and CFU-GEMM: rhuIL-3
(20 ng/mL), rhuGM-CSF (10 ng/mL), rhuEpo (3 U/mL), and rhuSCF (50 ng/mL). For CFU-GM, rhuGM-CSF (20 ng/mL), rhuIL-3 (20 ng/mL) and rhuSCF
(50 ng/mL) were added. For CFU-Mk, rhuIL-3 (5 ng/mL) was used as a
single growth factor. When transplanted NOD/SCID mouse BM cells were to
be evaluated for their human hematopoietic progenitor content, the FCS
in the methylcellulose medium was replaced with an equivalent volume of
a pretested pool of equivalently supportive normal human serum and
bovine plasma in the plasma clot assay was replaced with an equivalent
volume of human plasma. Plasma clot assays were adopted not only to
detect CFU-Mk colonies (with the addition of rhuIL-3), but for CFU-GM,
BFU-E, and CFU-GEMM as well (with the addition of rhuIL-3, rhuGM-CSF,
rhuSCF, and rhuEPO). G-CSF was omitted to minimize the stimulation of
murine clonogenic cells. These culture conditions have been reported to
be selective for colony formation by human progenitors and do not
support coexisting murine progenitors.8,13 In addition,
colonies grown in plasma-clot and colonies plucked from methylcellulose
cultures were stained with FITC-conjugated anti-human GPIIbIIIa, CD45,
CD13, and GpA and scored at the immunofluorescence microscope. The
presence of fluorescent colonies was the index of their human origin.
As a control, BM cells from untreated NOD/SCID mice were plated at identical cell concentrations in the same culture assays (plasma clot
and methylcellulose) containing only human serum and/or human plasma
and the above-reported human-specific growth factors. Dishes were
scored from day 12 up to day 21: in these culture conditions no
colonies could be detected.
LTC-IC.
The LTC-IC content of cell suspension was determined by limiting
dilution assays as previously described.27 Briefly, 10 to
1,000 purified CD34+ CB cells at the start of cultures or
suitable aliquots of cultured cells were washed and seeded onto
preestablished irradiated human BM stromal layers (derived by culturing
107 fresh BM MNC in a T25 flask for at least 2 weeks in 5 mL stromal medium [12.5% horse serum, 12.5% FCS, IMDM,
2-mercaptoethanol, 106 mol/L hydrocortisone, and
penicillin/streptomycin] and by plating the irradiated [15 Gy] and
trypsinized stroma at 7 × 103/cm2 in
24-well plates) and maintained at 37°C for 5 to 6 weeks with weekly
half media changes, at the end of which all cells were harvested and
plated for CFC determination in methylcellulose medium. These cultures
were incubated at 37°C for 2 weeks in the presence of 1.3%
methylcellulose, 30% FCS, EPO (3 U/mL),
IL-3 (20 ng/mL), G-CSF (20 ng/mL), GM-CSF (20 ng/mL), and SCF (50 ng/mL); LTC-IC enumeration was based on the number of CFU-C scored in the limiting dilution assay (LDA).
Stroma-free liquid cultures.
Stroma-free expansion cultures were performed as follows. (1) A total
of 10 to 20,000 CD34+ CB cells were cultured in
quadruplicate flat-bottomed 24-well plates in 1 mL of IMDM supplemented
with 10% pooled normal human serum with the following growth factors:
FL (50 ng/mL) + MGDF (20 ng/mL); SCF (50 ng/mL) + FL (50 ng/mL) + MGDF
(20 ng/mL); SCF (50 ng/mL) + FL (50 ng/mL) + MGDF (20 ng/mL) + IL-6 (10 ng/mL), which were added to each series of microwells twice a week. The wells were grown at 37°C. At initiation of the cultures, the number of CFC and CFU-Mk present in 1 mL of a single well was determined by
triplicate plasma clot assays. Every week all of the wells were
demidepopulated by removal of one half the culture volume (and cells),
which was replaced with fresh medium and growth factors. Cells of the
harvested media were counted and suitable aliquots of the cell
suspensions were assayed for CFC and CFU-Mk content, for
immunophenotype analysis (CD34+, CD34+
CD38) and for LTC-IC determination every 2 to 3 weeks by LDA. The total number of CFC or of LTC-IC generated CFC was
calculated as previously reported.27,28
(2) In a second series of expansion cultures, the same concentrations
of CD34+ CB cells were cultured in identical culture
conditions (same growth factors and serum). Cultures were set up in
quadruplicate. The only difference with the series (1) was that every
week, instead of being split in two, the cell suspension of each well
was resuspended in twice its volume, split in two, and plated in new
24-well plates. This way every week additional new 24-wells were set up
(eg, derived from the 1 mL prepared at start of cultures, 2, 4, and 8 wells were set up at weeks 2, 3, and 4, respectively).
(3) In this series of experiments, 1 to 5 × 104
CD34+cells/mL were deposited on the bottom of tissue
culture T25 or T75 flasks in quadruplicate.
Every week the culture volume was doubled. Cell counts were performed
every week. At weeks 4, 6, 8, and 12, the immunophenotype of the cells
harvested from the different sets of expansion was performed, and the
CFC content of each expansion set was determined by seeding suitable
aliquots of the pooled wells or flasks in triplicate plasma clot
cultures. For LTC-IC assay, limiting dilutions of the cell suspensions
deriving from each series of expansion wells or flasks were seeded onto
preirradiated stroma layers in 96-well plates for 5 weeks and then the
number of CFC generated by methylcellulose cultures was enumerated as described above.
To limit the volume of the expansion cultures, in most experiments, at
week 6, 7/8 of the total culture volume was cryopreserved and expansion
studies were performed with the remaining 1/8. When inocula were to be
prepared, 7/8 of week 6 cryopreserved cells were thawed,
washed, counted, and grown for an additional 6 to 10 days in the
presence of the same media and growth factors previously used; hence,
these inocula represented weeks 7 to 8 expanded cells. When weeks 9 to
10 and week 12 expanded cells were to be injected, the 1/8 expanded
cells (which had been cryopreserved at weeks 8 and 10, respectively)
were thawed, grown for an additional 6 to 10 days and mixed with 7/8 of
week 6 cryopreserved cells that had been thawed and grown as previously
described. The two samples were mixed, washed, and resuspended in a
small aliquot and injected in mice.
For limiting dilution studies, the 1/8 cells expanded for 6 and 8 weeks
(and then cryopreserved) were thawed and grown for an additional 6 to
10 days before being injected into mice. In these experiments, the
cells were not mixed with 7/8 of week 6 cryopreserved cells; hence they
represented the first dilution (1/8 of initial CD34+
cells). The next dilutions were prepared from the 1/8 cells (1/16, 1/32, etc).
DNA extraction and analysis of human cell engraftment.
High molecular weight DNA was extracted from the BM of transplanted
mice by phenol-chloroform extraction using standard protocols. DNA was
digested with EcoRI and separated by agarose gel electrophoresis, transferred onto a positively charged nylon membrane, and probed with a
labeled human chromosome 17-specific -satellite probe (p17H8) (limit
of detection, approximately 0.05% human DNA). To quantify the level of
human cell engraftment, the intensity of the characteristic 2.7-kb band
in samples was compared with those of human: mouse DNA control mixtures
(0%, 0.1%, 1%, 10% human DNA).
Statistical analysis.
For purposes of LDAs (LTC-IC and SRC), Poisson statistics for the
single-hit model were applied. The frequency of LTC-IC and SRC in cell
suspensions was calculated using maximum likelihood estimator.32
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RESULTS |
Groups of sublethally irradiated NOD/SCID mice were injected with
decreasing concentrations of CD34+ CB cells, which had been
previously separated from several CB samples, then pooled, and stored
frozen until the time of transplant. Six to 8 weeks after inoculation,
BM cells of the sacrificed animals were obtained from both femurs and
both tibias and assessed for the presence of human hematopoietic cells.
Table 1 presents a summary of these data. While 2 × 105 and 1 × 105 CD34+
cells engrafted the totality of mice, 35,000 and 20,000 CD34+ cells engrafted 4 of 6 and 3 of 7 mice, respectively.
No mouse was found to be engrafted by 10,000 CD34+ cells.
The level of engraftment was variable and dependent on the number of
injected CD34+ cells (human CD45+,
CD71+, and -GpA+ cells ranged from 0.18% to
25%). The level of human engraftment was also evaluated by DNA
analysis (as shown in Fig 1). The frequency of SRC in
the unmanipulated cryopreserved CD34+ CB cells was found to
be 1 in 30,900 CD34+ cells (95% confidence interval
1:53,600 to 1:17,800). It was similar to that of fresh
CD34+ CB cells (1 in 29,800; 95% confidence interval
1:51,700 to 1:17,300). BM cells of engrafted mice were further analyzed
for evidence of multilineage development from input CD34+
cells. Cells within the huCD45 gate or costained with CD45 were quantified for myeloid and lymphoid surface markers as well as for the
expression of the CD34 antigen. Erythroid and megakaryocyte surface
marker expression was also investigated. Figure 2A and B
shows representative analysis of BM cells from engrafted and nonengrafted mice. As an additional proof of human myeloid engraftment, BM cells of sacrificed mice were cultured in semisolid assays in
culture conditions that have been reported to allow human and not mouse
colony growth.8,13 In addition, colonies were stained with
MoAbs recognizing human total leukocytes, granulocyte- macrophages, erythroid cells, and megakaryocytes and only fluorescent colonies (consisting of human cells) were counted (Table 1).
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Table 1.
Main Characteristics of Injected CB CD34+
Cells and Their Ability to Engraft the BM of NOD/SCID Recipients
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| Fig 1.
Representative DNA analysis of human cell engraftment in
the BM of NOD/SCID mice transplanted with 1 × 104, 2 × 104, and 3.5 × 104 unmanipulated
CD34+ CB cells. Cells were injected together with
preirradiated 2 × 105 CD34 CB cells as
carrier cells. Human DNA was assessed by Southern analysis using a
human chromosome 17-specific  -satellite probe. Human:mouse controls
are given as percent human DNA.
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| Fig 2.
(A) Representative fluorescence-activated cell sorting
(FACS) profiles of marrow cells from NOD/SCID mice transplanted 8 weeks
previously with unmanipulated CD34+ CB cells. (a)
Negative control: a nonengrafted mouse (transplanted with 2 × 105 irradiated CD34 CB carrier cells).
(Middle) A mouse transplanted with 1 × 105
CD34+ CB cells. (Bottom) A mouse transplanted with 2 × 104 CD34+ cells. CD45/CD34 and CD45/CD19
analysis was performed on total BM cells. (B) Multilineage engraftment
in the BM of a representative mouse transplanted with 1 × 105 unmanipulated CD34+ CB cells. Analysis of
lineage markers (CD45/CD34, CD19, CD41, and CD13/CD33) was performed on
cells comprised within the CD45 gate. Analysis of GpA/CD71-positive
cells was performed on total BM cells.
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In the second part of our study, CB CD34+ cells were
cultured in vitro in stroma-free liquid cultures as
described.27,28 Cell counts, CD34+,
CD34+38 subpopulations, CFC output, and
LTC-IC expansion obtained in the different settings were compared. At
weeks 4, 6, and 8 of expansion cultures, all cells were harvested from
quadruplicate cultures, counted, and subjected to the same screening
(immunophenotype analysis, CFC output, and LTC-IC enumeration) as
described before. All of the remaining cells (in 7.5 to 127 mL) were
washed, cryopreserved, and stored frozen for further injection into
sublethally irradiated NOD/SCID recipients (one mouse for each expansion).
Evident from these experiments is the finding that only marginal
differences could be detected in the qualitative and quantitative content of the three different expansion procedures
(Table 2). After a 4-week expansion in the presence of
FL+MGDF, cell counts ranged from 148-fold to 240-fold the initial
number; with SCF+FL+MGDF, the cell count was 444-fold to 515-fold the
input cells; the four-factor combination appeared also most effective
in inducing and maintaining a sustained cell production (348-fold to
587-fold) and CFC output (55-fold to 80-fold). Expansion of the LTC-IC
population was induced to a very similar degree by all three growth
factor combinations and in all three culture settings (Table 2). Also,
at weeks 6 and 8 of expansion cultures, cell number, percent of
CD34+ cells, as well as CFC production and LTC-IC
expansion, were not much different if demidepopulated and expanded
wells or flasks were compared (Table 2). Table 3 shows
the proportion of different hematopoietic subpopulations at start of
cultures and at different time points of expansion. No differences
could be detected in the cellular composition of the cells expanded
with the three growth factor combinations (not shown).
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Table 3.
Antigenic Composition of CD34+ CB Cells at
Initiation of Liquid Cultures and at Various Time Intervals During Ex
Vivo Expansion
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To determine whether the expanded cells retained their ability of full
hematopoietic reconstitution (ie, sustained and multilineage engraftment) cohorts of 6- to 8-week-old NOD/SCID mice were irradiated with 350 to 375 cGy from a 137Cs source and, within 24 hours, injected with either 35,000, 20,000, or 10,000 CD34+
CB cells, which were cryopreserved at the start of cultures, or the
progeny of identical numbers of CD34+ cells that were grown
for 4 weeks with FL+MGDF, SCF+FL+MGDF, IL-6+SCF+FL+MGDF (one mouse for
each expansion). Six to 8 weeks later, BM cells of all mice were
prepared, counted, and subjected to DNA and phenotype analysis to
determine whether or not human cells were present; which subpopulation
was represented and whether human hematopoietic progenitors (BFU-E,
CFU-GM, CFU-GEMM) could be detected. Table 4 shows the
results of three separate experiments performed in quadruplicate.
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Table 4.
Comparison Between Cell Number, CFC, and LTC-IC Injected
and Ability to Engraft the BM and the Spleen of NOD/SCID Mice by
Human CB Cells Cultured in Stroma-Free Cultures for 4 Weeks
|
|
In keeping with the results reported in Table 1, only 2 of 6 mice that
had been injected with 20,000 or 10,000 cryopreserved uncultured
CD34+ CB cells were engrafted and the level of human
engraftment was very low. These data were confirmed by subsequent DNA
analysis: the results obtained by injecting an additional 13 mice with
35,000 and 20,000 unmanipulated CD34+ cells are shown in
Fig 3. The same figure, Fig 4 and Table
4 show that, at the opposite side, mice injected with the cells that
had been generated by the same number of initial CD34+ CB
cells in 4-week expansion cultures supported by FL+MGDF, SCF+FL+MGDF, and IL6+SCF+FL+MGDF engrafted the vast majority (15 of 18) of NOD/SCID
mice, although at a variable degree. Human CD45+,
CD71+, and GpA+ cells constituted 6% to 23%
of the entire BM. Further analysis of the engrafted BM cells showed the
consistent presence of human CD45+CD19+ and
CD45+CD34+ cells. CD33+,
CD41+, CD71+, and GpA+ cells were
also represented (not shown). Plasma clot assays performed by seeding
BM cells of transplanted NOD/SCID mice showed that human CFU-GM, BFU-E,
CFU-GEMM, and CFU-Mk were indeed present (Table 4). Colonies grown in
plasma-clot assays were scored at the immunofluorescent microscope
after being stained with FITC-conjugated anti-human CD41, CD45, CD13,
and -GpA antibodies. All of the colonies were positive, thus
indicating their human origin.
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| Fig 3.
Engraftment of human CB CD34+cells at start
of cultures and of their progeny after 4 to 12 weeks of expansion. The
level of human engraftment was evaluated by flow cytometry by
determining the percent of human CD45+,
CD71+, and GpA+ cells within the total BM
cells in individual NOD/SCID mice. Each animal was injected either with
unmanipulated CD34+ CB cells (2 × 104,  ;
3.5 × 104,  ) either with the cells generated by
initial 2 × 104 or 3.5 × 104
CD34+ cells after 4, 7 to 8, 9 to 10 or 12 weeks of
cultures as described in Materials and Methods. Each circle represents
an individual mouse.
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| Fig 4.
Representative Southern blot analysis of individual
NOD/SCID mice transplanted with 1 × 104, 2 × 104, and 3.5 × 104 unmanipulated
CD34+ CB cells and with week 4 expanded cells (deriving
from 1 × 104 initial CD34+ cells). DNA was
extracted from the murine BM at week 8 after transplant and hybridized
with a human chromosome 17-specific -satellite probe.
|
|
Expansion cultures were then extended for up to 12 weeks. Flow
cytometry analysis of the cells harvested at different time points did
not show substantial differences in the CD34 expression of the cells
grown in the presence of the three growth factor combinations.
CD34+ cells still constituted a good proportion of the
total cell population ( Fig 5). CD34+
CD38 cells were also detected (Fig
6).
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| Fig 5.
Analysis of CD34 antigen expression on CB
CD34+ cells at start of cultures (week 0) and at weeks 4, 6, 8, and 12 of ex vivo expansion in stroma-free cultures supplemented
with FL+ MGDF and IL6+SCF+FL+MGDF.
|
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| Fig 6.
Analysis of CD34 and CD38 antigen expression on
CD34+ CB cells at start of cultures (week 0)
and after 4 and 8 weeks of expansion in stroma-free cultures
supplemented with FL+ MGDF and IL6+SCF+FL+MGDF.
|
|
Cells cultured for 6 to 7 weeks engrafted the vast majority of injected
mice (Fig 3). Both DNA analysis and flow cytometry performed on BM
cells of the transplanted mice showed that human cells were indeed
detectable. CD45+, CD71+, and GpA+
cells accounted for up to 20% of the entire BM after 7 to 8 weeks of
liquid cultures; human engraftment was detectable at fairly good levels
(up to 10%) in mice injected with cells expanded for up to 12 weeks.
Further flow cytometry analysis of the BM cells of the engrafted mice
showed that all of the different hematopoietic lineages were
represented (Fig 7A and B). In addition, human colonies were detected in semisolid cultures prepared with BM cells of mice
transplanted with expanded CB cells (Table 5). Expansion studies performed beyond the week 6 cryopreservation comprised only one
eighth of the initial CD34+ cells; this is why we first
inoculated the expanded 1/8 together with the cryopreserved 7/8 (hence
the engraftment activity might be due completely to the latter).
Therefore, to better define whether weeks 7 to 8 and week 10 expanded
cells contained SRC and to quantitate the extent of the amplification
of the in vivo repopulating cells in the expansion cultures, the 1/8
cells expanded for 7 to 8 and 9 to 10 weeks were injected into
sublethally irradiated NOD/SCID recipients without further mixing with
the remaining 7/8 and were considered to represent the first dilution.
Taking into account the number of the CD34+ cells at the
start of cultures (50,000, 35,000, 20,000, or 10,000 CD34+
cells), the number of the initial cells injected per mouse was calculated (eg, 1/8 of the cells expanded by 20,000 initial
CD34+ cells represents the corresponding initial 2,500 CD34+ cells; 1/16 of initial 20,000 cells represents 1,250 cells, and so on). Shown in Table 5 are the results of two separate
experiments performed in quadruplicate and injected in replicate mice
(two per cell dose). BM cells of the injected recipients contained human cells. The degree of human engraftment in mice injected with
cells expanded for 7 to 8 weeks ranged between 6.8% and 18%. In
particular, BM cells of engrafted mice when cultured in semisolid assays specific for human progenitors, generated colonies (CFU-GM, CFU-Mk, and BFU-E), which were positively stained by human specific MoAbs. The experiments reported in Table 5 and additional limiting dilution experiments reported in Fig 8 showed that the
cells generated after 7 to 8 weeks of expansion by initial 1,250, 625, and 312 CD34+ cells repopulated 9 of 10, 7 of 10, and 8 of
14 mice, respectively. Also, Figs 9 and
10 show that after 9 to 10 weeks of expansion, the
cells obtained by initial 1,560 CD34+ cells engrafted 7 of
7 mice, those obtained by 625 CD34+ cells engrafted 5 of 7 mice, and those deriving from 312 CD34+ cells engrafted 7 of 12 mice.
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| Fig 7.
(A) Representative FACS profiles of marrow cells from
individual NOD/SCID mice transplanted 8 weeks previously with cells
deriving from 3.5 × 104 CD34+ CB cells
after a 10-week expansion in stroma-free cultures containing IL-6, SCF,
FL and MGDF. From top to bottom: isotype control and (a) a nonengrafted
mouse. Human CD45/CD34 and CD45/CD19 in the BM cells of a mouse
transplanted with 1/8, 1/25, and 1/70 of the cell progeny deriving from
3.5 × 104 initial CD34+ cells. CD45/CD34
and CD45/CD19 analysis was performed on total BM cells. (B)
Multilineage engraftment in the BM of a representative mouse
transplanted with week 10 expanded cells (deriving from 2 × 104 initial CD34+ cells). Analysis of lineage
markers (CD45/CD34, CD19, CD41, and CD13/CD33) was performed on cells
comprised within the CD45 gate. Analysis of GpA/CD71 cells was
performed on total BM cells.
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View this table:
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Table 5.
Determination of the Cell Number, CFC, LTC-IC, and the
Ability to Engraft the BM of NOD/SCID Recipients by Human CB Cells
During Stroma-Free Cultures
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| Fig 8.
Summary of the level of human cell engraftment in the BM
of 35 mice transplanted with cells deriving from ex vivo expansion
cultures at weeks 7 to 8 in the presence of IL-6, SCF, FL, and MGDF.
Individual NOD/SCID mice (each symbol represents a mouse) were injected
8 weeks previously with fractions of the expanded cells, corresponding
to numbers of initial CD34 cells indicated on the abscissa. The level
of human engraftment in the mouse BM was evaluated by both flow
cytometry (as percent of human CD45, GpA, and CD71 positive cells) and
DNA analysis as described in Materials and Methods.
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| Fig 9.
Summary of the level of human cell engraftment in the BM
of 35 mice transplanted 8 weeks previously with cells deriving from ex
vivo expansion cultures at weeks 9 to 10 in the presence of IL-6, SCF,
FL, and MGDF. Individual NOD/SCID mice (each symbol represents a mouse)
were injected with fractions of the expanded cells, corresponding to
numbers of initial CD34+ cells indicated on the abscissa.
The level of human engraftment in the mouse BM was evaluated by both
flow cytometry and DNA analysis as described in Materials and
Methods.
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| Fig 10.
Representative Southern blot analysis of individual
NOD/SCID mice transplanted with expanded cells from replicate flasks at
week 10 of expansion. Quadruplicate cultures were initiated with 2 × 104 CD34+ CB cells. After 10 weeks of
expansion, 2 mice were injected with the corresponding progeny of
initial CD34+ cells (1 mouse per expansion) lanes 8 to 9;
2 mice with 1/12 of the expanded cells (corresponding to initial 1,560 CD34+ cells) lanes 6 to 7; 2 mice with 1/32 (=625
initial cells) lanes 4 to 5; and 2 mice with 1/64 (=312 initial
cells) lanes 2 to 3. Lane 1: A nonengrafted mouse (inoculated with 2 × 104 CD34+ cells at start of cultures,
plus 2 × 105 CD34 irradiated CB cells).
|
|
Poisson statistics allowed us to calculate that the frequency of SRC
after 7 to 8 weeks of expansion was 1 in 471 initial CD34+
cells (95% confidence interval 1:571 to 1:296), while, after 9 to 10 weeks, it was 1 in 393 initial CD34+ cells (95% confidence
interval 1:678 to 1:228).
| |
DISCUSSION |
The development of SRC assay for primitive human hematopoietic cells
capable of repopulating the BM of NOD/SCID immune deficient mice with
myeloid and lymphoid lineages provides a new approach to investigate
the organization of the human hematopoietic system and to characterize
primitive stem cells. The experiments reported here show that CB
CD34+ cells after a 4- to 10-week expansion in stroma-free
liquid cultures containing the combinations of a few growth factors
(FL+MGDF±SCF±IL-6) retained their capacity to completely
engraft the BM of sublethally irradiated NOD/SCID recipients. The level
of engraftment, well above that usually observed when the same number
of uncultured cells were injected in the same recipients, suggested
that SRC were not only maintained, but, rather, expanded.
This result supports and extends our previous findings that
CD34+ CB cells could be grown in vitro for an extremely
long period of time, during which a massive and continuously increasing
production of CD34+, CD34+
CD38 cells, committed unipotent and multipotent
progenitors occurred; also primitive stem cells, identified in vitro as
LTC-IC were shown to be amplified more than 200,000-fold after 20 weeks. It was concluded that the extremely prolonged and impressive
expansion of progenitors belonging to all of the hematopoietic lineages was supported by a similar expansion of primitive progenitors and that
in our system, some degree of self-renewal, beside differentiation, was
taking place.27,28
The culture conditions used in the present study were similar to those
previously reported and yielded similar numbers of CFC and LTC-IC,
attesting that the small-scale demidepopulation assay could be
reproduced in a larger scale expansion setting without losing its
initial quality. In addition, in the cultures presented here, 10%
human serum was substituting for 10% FCS, therefore a further step
toward a clinical application has been secured.
In the first part of our study when decreasing numbers of uncultured CB
CD34+ cells were injected into sublethally irradiated
NOD/SCID recipients, less than 35,000 CD34+ cells did not
engraft the totality of the recipients. Using Poisson statistics,33 the frequency of SRC in the cryopreserved
CD34+ population was calculated to be 1 in 30,900, while in
fresh CD34+ cells, it was 1 in 29,800, which appears in
keeping with that reported by others.14,16,33 Four to 10 weeks of culturing CD34+ CB cells in stroma-free cultures
led to an increase in the repopulating capacity of the cultured
hematopoietic cells. Injection of the cultured cells deriving from
35,000, 20,000, or 10,000 initial CD34+ cells resulted in a
significant increase in the level of human engraftment, similar to that
observed by injecting at least 100,000 uncultured CD34+ cells.
Limiting dilution experiments performed by injecting NOD/SCID mice with
decreasing concentrations of cells expanded for up to 10 weeks allowed
us to show that the frequency of SRCs after 7 to 8 weeks of expansion
was 1 in 471 and after 9 to 10 weeks, it was 1 in 391 input
CD34+ cells, therefore the expansion of SRC was 65-fold and
78-fold, respectively, 60-fold and 70-fold if compared with the
frequency of SRC in fresh CD34+ CB cells. Recently, it has
been reported that incubation of CD34+
CD38 CB cells in serum-free medium containing FL,
SCF, IL-3, IL-6, and G-CSF for 5 to 8 days, resulted in a 100-fold
expansion of CFC, a 4-fold expansion of LTC-IC, and a modest (2-fold),
but significant, increase of CRU. CRU were found, although at different frequencies, also in the CD34+ CD38+ CB
subpopulations and with a distribution between the two CB subsets as
LTC-IC. It was concluded that conditions, which were able to stimulate
LTC-IC expansion, might also stimulate increases in CRU.16
Similarly, Bhatia et al29 showed a fourfold increase of SRC
after a 4-day incubation of CD34+ CD38
CB cells in stroma-free conditions; SRC, however, were lost after 9 days of culture. Gan et al30 reported that cocultures of BM or CB cells on allogeneic stroma layers for up to 3 weeks resulted in a
decrease and a final loss of in vivo repopulating ability in NOD/SCID
mice recipients; in contrast to the loss of SRC, the cultured cells
frequently contained an equal or higher number of LTC-IC compared with
the fresh cells. Additionally, while this report was being revised, it
was reported that human CD34+ CD38 BM
cells, after 6-day stroma-free suspension cultures containing FL, TPO,
and SCF retained their in vivo repopulating capacity in the SCID/hu
bone assay.34 Also, Xu et al31 recently showed that CB cells could maintain their in vivo repopulating ability after
at least 4 weeks of coculture on a stromal cell line derived from the
aorta-gonad-mesonephros region of mouse embryo.
Cell populations, culture conditions (stromal cocultures or stroma-free
cultures), as well as the growth factors used in the various studies
are different, therefore the somewhat different results cannot be
compared. However, it is becoming increasingly evident that primitive
stem cells, defined by their ability to completely engraft a
myeloablated recipient, can be maintained in vitro for long periods of
time and are likely to undergo self-renewal divisions.
A very controversial issue is the role played by stroma cocultures:
even though the presence of BM stroma layers has been shown to increase
the frequency of gene transfer into primitive cells, compared with
using viral supernatants and to prevent the loss of stem cell quality
during ex vivo expansion of peripheral blood stem
cells,35-37 other reports suggest that the long-term repopulating ability of cultured cells decreases using BM stroma cocultures.38,39
The vast majority of prior studies aimed at developing clinical
applications of expansion protocols have adopted culture conditions that resulted in a marked expansion of cell counts, CD34+
cells, CFC, and even LTC-IC in a short period of time. However, the
expansion was transient, soon followed by a rapid decline of cell
number, of CFC output, and disappearance of LTC-IC, which indicated the
exhaustion of the stem cell pool. In our culture system, it is possible
to obtain very large numbers of cells and progenitors belonging to the
more mature hematopoietic compartments and, at the same time, to
maintain and even expand several-fold the primitive in vivo
repopulating stem cells. This information could prove essential to
design and test conditions for ex vivo activation and expansion of
immature hematopoietic cells and for various experimental purposes,
such as required for the development of efficient gene transfer
protocols into hematopoietic cells with retention of repopulating ability.
| |
FOOTNOTES |
Submitted May 11, 1998; accepted January 29, 1999.
Supported by grants from Associazione Italiana per la Ricerca sul
Cancro (AIRC; Milan, Italy) and from the Ministero
dell'Università e della Ricerca Scientifica e Tecnologica
(MURST) (to W.P. and M.A.). A.D. is a recipient of the FIRC grant; A.S.
and L.G. are both recipients of the "G. Ghirotti Foundation,"
sez. Piemonte grants.
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 Wanda Piacibello, MD, Department of
Biomedical Sciences and Human Oncology, Clinical Section, Via Genova 3, 10126 Torino, Italy; e-mail:
w.piacibello{at}mail.ircc.unito.it.
| |
REFERENCES |
1.
Spangrude G, Smith L, Uchida N, Ikuta F, Heimfeld S, Friedman J, Weissman IL:
Mouse hematopoietic stem cells.
Blood
78:1395, 1991[Free Full Text]
2.
Ogawa M:
Differentiation and proliferation of hematopoietic stem cells.
Blood
81:2844, 1993[Abstract/Free Full Text]
3.
Berardi AC, Wang A, Levine JD, Lopez P, Scadden DT:
Functional isolation and characterization of human hematopoietic stem cells.
Science
267:104, 1995[Abstract/Free Full Text]
4.
To LB, Haylock DN, Simmons PJ, Juttner CA:
The biology and clinical uses of blood stem cells.
Blood
89:2233, 1997[Free Full Text]
5.
Sutherland HJ, Lansdorp PM, Henkelman DH, Eaves AC, Eaves CJ:
Functional characterization of individual human hematopoietic cells cultured at limiting dilution on supportive marrow stromal layers.
Proc Natl Acad Sci USA
87:3584, 1990[Abstract/Free Full Text]
6.
Hao QL, Shah AJ, Thienmann FT, Smogorzewska EM, Crooks GM:
A functional comparison of CD34+CD38 cells in cord blood and bone marrow.
Blood
86:3745, 1995[Abstract/Free Full Text]
7.
Kamel-Reid S, Dick JE:
Engraftment of immune-deficient mice with human hematopoietic stem cells.
Science
242:1706, 1988[Abstract/Free Full Text]
8.
Lapidot T, Pflumio F, Doedens M, Murdoch B, Williams DE, Dick JE:
Cytokine stimulation of multilineage hematopoiesis from immature human cells engrafted into SCID mice.
Science
255:1137, 1992[Abstract/Free Full Text]
9.
Kyoizumi S, Baum CM, Kaneshima H, McCune JM, Yee EJ, Namikawa R:
Implantation and maintenance of functional human bone marrow in SCID-hu mice.
Blood
79:1704, 1992[Abstract/Free Full Text]
10.
Nolta JA, Hanley MB, Kohn DB:
Sustained human hematopoiesis in immunodeficient mice by cotransplantation of marrow stroma expressing human interleukin-3: Analysis of gene transduction of long-lived progenitors.
Blood
83:3041, 1994[Abstract/Free Full Text]
11.
Lowry PA, Shultz LD, Greiner DL, Hesselton RM, Kittler ELW, Tiarks CY, Rao SS, Reilly J, Leif JH, Ramshaw H, Stewart FM, Quesenberry PJ:
Improved engraftment of human cord blood cells in NOD/LtSz-scid/scid mice after irradiation or multiple-day injections into unirradiated recipients.
Biol Blood Marrow Transplant
2:15, 1996[Medline]
[Order article via Infotrieve]
12.
Pflumio F, Izac B, Katz A, Shultz LD, Vainchenker W, Coulombel L:
Phenotype and function of human hematopoietic cells engrafting immune-deficient CB17-severe combined immunodeficiency mice and nonobese diabetic-severe combined immunodeficiency mice after transplantation of human cord blood mononuclear cells.
Blood
88:3731, 1996[Abstract/Free Full Text]
13.
Cashman JD, Lapidot T, Wang JCY, Doedens M, Shultz LD, Lansdorp P, Dick JE, Eaves CJ:
Kinetic evidence of the regeneration of multi-lineage hematopoiesis from primitive cells in normal human bone marrow transplanted into immunodeficient mice.
Blood
89:4307, 1997[Abstract/Free Full Text]
14.
Hogan CJ, Shpall EJ, McNulty O, McNiece I, Dick JE, Shultz LD, Keller G:
Engraftment and development of human CD34+-enriched cells from umbilical cord blood in NOD/LtSz-scid/scid Mice.
Blood
90:85, 1997[Abstract/Free Full Text]
15.
Dick JE:
Normal and leukemic human stem cells assayed in SCID mice.
Semin Immunol
8:197, 1996[Medline]
[Order article via Infotrieve]
16.
Conneally E, Cashman J, Petzer A, Eaves C:
Expansion in vitro of transplantable human cord blood stem cells demonstrated using a quantitative assay of their lympho-myeloid repopulating activity in nonobese diabetic-scid/scid mice.
Proc Natl Acad Sci USA:
94:9836, 1997[Abstract/Free Full Text]
17.
Larochelle A, Vormoor J, Hanenberg H, Wang JCY, Bhatia M, Lapidot T, Moritz T, Murdoch B, Xiao XL, Kato I, Williams DA, Dick JE:
Identification of primitive human hematopoietic cells capable of repopulating NOD/SCID mouse bone marrow: Implications for gene therapy.
Nat Med
2:1329, 1996[Medline]
[Order article via Infotrieve]
18.
Bhatia M, Wang JCY, Kapp U, Bonnet D, Dick JE:
Purification of primitive human hematopoietic cells capable of repopulating immune-deficient mice.
Proc Natl Acad Sci USA
94:5320, 1997[Abstract/Free Full Text]
19.
Cashman J, Bockhold K, Hogge DE, Eaves AC, Eaves CJ:
Sustained proliferation, multi-lineage differentiation and maintenance of primitive human hemopoietic cells in NOD/SCID mice transplanted with human cord blood.
Br J Haematol
97:1026, 1997
20.
Gluckman E, Broxmeyer HE, Auerbach AD, Friedman H, Douglas G, Devergie A, Esperou H, Thierry D, Socie G, Lehn P, Cooper S, English D, Kurtzberg J, Bard J, Boyse E:
Hematopoietic reconstitution in a patient with Fanconi's anemia by means of umbilical cord blood from an HLA-identical sibling.
N Engl J Med
321:1174, 1989[Medline]
[Order article via Infotrieve]
21.
Broxmeyer HE, Gluckman E, Auerbach AD, Douglas GW, Friedman H, Cooper S, Hangoc G, Kurtzberg J, Bard J, Boyse EA:
Human umbilical cord blood: A clinically useful source of transplantable hematopoietic stem/progenitor cells.
Int J Cell Cloning
8:76, 1990
22.
Hows JM, Bradley BA, Marsh JCW, Luft T, Coutinho L, Testa NG, Dexter TM:
Growth of umbilical cord blood in longterm haemopoietic cultures.
Lancet
340:73, 1992[Medline]
[Order article via Infotrieve]
23.
Lu L, Xiao M, Shen R-N, Grigsby S, Broxmeyer HE:
Enrichment, characterization and responsiveness of single primitive CD34+++ human umbilical cord blood hematopoietic progenitors with high proliferative and replating potential.
Blood
81:41, 1993[Abstract/Free Full Text]
24.
Cairo MS, Wagner JE:
Placental and/or umbilical cord blood: An alternative source of hematopoietic stem cells for transplantation.
Blood
90:4665, 1997[Free Full Text]
25.
Petzer AL, Hogge DE, Lansdorp PM, Reid DS, Eaves CJ:
Self-renewal of primitive human hematopoietic cells (long-term-culture-initiating cells) in vitro and their expansion in defined medium.
Proc Natl Acad Sci USA
93:1470, 1996[Abstract/Free Full Text]
26.
Petzer AL, Zandstra PW, Piret JM, Eaves CJ:
Differential cytokine effects on primitive (CD34+CD38-) human hematopoietic cells: Novel responses to Flt3-ligand and thrombopoietin.
J Exp Med
183:2551, 1996[Abstract/Free Full Text]
27.
Piacibello W, Sanavio F, Garetto L, Severino A, Bergandi D, Ferrario J, Fagioli F, Berger M, Aglietta M:
Extensive amplification and self-renewal of human primitive hematopoietic stem cells from cord blood.
Blood
89:2644, 1997[Abstract/Free Full Text]
28.
Piacibello W, Sanavio F, Garetto L, Severino A, Danè A, Gammaitoni L, Aglietta M:
Differential growth factor requirement of primitive cord blood hematopoietic stem cell for self-renewal and amplification vs proliferation and differentiation.
Leukemia
12:718, 1998[Medline]
[Order article via Infotrieve]
29.
Bhatia M, Bonnet D, Kapp U, Wang CY, Murdoch B, Dick JE:
Quantitative analysis reveals expansion of human hematopoietic repopulating cells after short-term ex vivo culture.
J Exp Med
186:619, 1997[Abstract/Free Full Text]
30.
Gan OI, Murdoch B, Larochelle A, Dick JE:
Differential maintenance of primitive human SCID-repopulating cells, clonogenic progenitors, and long-term culture-initiating cells after incubation on human bone marrow stromal cells.
Blood
90:641, 1997[Abstract/Free Full Text]
31.
Xu M, Tsuji K, Ueda T, Mukouyama Y, Hara T, Yang F, Ebihara Y, Matsuoka S, Manabe A, Kikuchi A, Ito M, Miyajima A, Nakahata T:
Stimulation of mouse and human primitive hematopoiesis by murine embryonic aorta-gonad-mesonephros-derived stromal cell lines.
Blood
92:3032, 1998
32.
Taswell C:
Limiting dilution assays for the determination of immunocompetent cell frequencies: I. Data analysis.
J Immunol
26:1614, 1981
33.
Wang JCY, Doedens M, Dick JE:
Primitive human hematopoietic cells are enriched in cord blood compared to adult bone marrow or mobilized peripheral blood as measured by the quantitative in vivo SCID-repopulating cell (SRC) assay.
Blood
89:3924, 1997
34.
Luens KM, Travis MA, Chen BP, Hill BL, Scollay R, Murray LJ:
Thrombopoietin, kit ligand, flk2/flt3 ligand together induce increased numbers of primitive hematopoietic progenitors from human CD34+ Thy-1+ Lin- cells with preserved ability to engraft SCID-hu bone.
Blood
91:1206, 1998[Abstract/Free Full Text]
35.
Moore KA, Deisseroth AB, Reading CL, Williams DE, Belmont JW:
Stromal support enhances cell-free retroviral vector transduction of human bone marrow long-term culture-initiating cells.
Blood
79:1393, 1992[Abstract/Free Full Text]
36.
Nolta JA, Smogorzewska EM, Kohn DB:
Analysis of optimal conditions for retroviral-mediated transduction of primitive human hematopoietic cells.
Blood
86:101, 1995[Abstract/Free Full Text]
37.
Breems DA, Blokland EAW, Siebel KE, Mayen AEM, Engels LJA, Ploemacher RE:
Stroma-contact prevents loss of hematopoieticstem cell quality during ex vivo expansion of CD34+ mobilized peripheral blood stem cells.
Blood
91:111, 1998[Abstract/Free Full Text]
38.
Harrison DE, Lerner CP, Spooncer E:
Erythropoietic repopulating ability of stem cells from long-term marrow culture.
Blood
69:1021, 1997[Abstract/Free Full Text]
39.
Van der Sluijs JP, van den Bos C, Baert MRM, van Beurden CAJ, Ploemacher RE:
Loss of long-term repopulating ability in long-term bone marrow culture.
Leukemia
7:725, 1993[Medline]
[Order article via Infotrieve]
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4326 - 4333.
[Abstract]
[Full Text]
[PDF]
|
|
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|
|
|
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P. Ioannidis, L. G. Mahaira, S. A. Perez, A. D. Gritzapis, P. A. Sotiropoulou, G. J. Kavalakis, A. I. Antsaklis, C. N. Baxevanis, and M. Papamichail
CRD-BP/IMP1 Expression Characterizes Cord Blood CD34+ Stem Cells and Affects c-myc and IGF-II Expression in MCF-7 Cancer Cells
J. Biol. Chem.,
May 20, 2005;
280(20):
20086 - 20093.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. K. Ballen
New trends in umbilical cord blood transplantation
Blood,
May 15, 2005;
105(10):
3786 - 3792.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. De Felice, C. Tatarelli, M. G. Mascolo, C. Gregorj, F. Agostini, R. Fiorini, V. Gelmetti, S. Pascale, F. Padula, M. T. Petrucci, et al.
Histone Deacetylase Inhibitor Valproic Acid Enhances the Cytokine-Induced Expansion of Human Hematopoietic Stem Cells
Cancer Res.,
February 15, 2005;
65(4):
1505 - 1513.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. Yang, I. Dybedal, D. Bryder, L. Nilsson, E. Sitnicka, Y. Sasaki, and S. E. W. Jacobsen
IFN-{gamma} Negatively Modulates Self-Renewal of Repopulating Human Hemopoietic Stem Cells
J. Immunol.,
January 15, 2005;
174(2):
752 - 757.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. P. Chute, G. G. Muramoto, J. Fung, and C. Oxford
Soluble factors elaborated by human brain endothelial cells induce the concomitant expansion of purified human BM CD34+CD38- cells and SCID-repopulating cells
Blood,
January 15, 2005;
105(2):
576 - 583.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. J. Schuringa, K. Y. Chung, G. Morrone, and M. A.S. Moore
Constitutive Activation of STAT5A Promotes Human Hematopoietic Stem Cell Self-Renewal and Erythroid Differentiation
J. Exp. Med.,
September 7, 2004;
200(5):
623 - 635.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. Gammaitoni, K. C. Weisel, M. Gunetti, K.-D. Wu, S. Bruno, S. Pinelli, A. Bonati, M. Aglietta, M. A. S. Moore, and W. Piacibello
Elevated telomerase activity and minimal telomere loss in cord blood long-term cultures with extensive stem cell replication
Blood,
June 15, 2004;
103(12):
4440 - 4448.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. Rollini, S. Kaiser, E. Faes-van't Hull, U. Kapp, and S. Leyvraz
Long-term expansion of transplantable human fetal liver hematopoietic stem cells
Blood,
February 1, 2004;
103(3):
1166 - 1170.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
F. Mazurier, O. I. Gan, J. L. McKenzie, M. Doedens, and J. E. Dick
Lentivector-mediated clonal tracking reveals intrinsic heterogeneity in the human hematopoietic stem cell compartment and culture-induced stem cell impairment
Blood,
January 15, 2004;
103(2):
545 - 552.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
F. Leone, E. Perissinotto, G. Cavalloni, V. Fonsato, S. Bruno, N. Surrenti, D. Hong, A. Capaldi, M. Geuna, W. Piacibello, et al.
Expression of the c-ErbB-2/HER2 proto-oncogene in normal hematopoietic cells
J. Leukoc. Biol.,
October 1, 2003;
74(4):
593 - 601.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. Jaroscak, K. Goltry, A. Smith, B. Waters-Pick, P. L. Martin, T. A. Driscoll, R. Howrey, N. Chao, J. Douville, S. Burhop, et al.
Augmentation of umbilical cord blood (UCB) transplantation with ex vivo-expanded UCB cells: results of a phase 1 trial using the AastromReplicell System
Blood,
June 15, 2003;
101(12):
5061 - 5067.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. E. Broxmeyer, E. F. Srour, G. Hangoc, S. Cooper, S. A. Anderson, and D. M. Bodine
High-efficiency recovery of functional hematopoietic progenitor and stem cells from human cord blood cryopreserved for 15 years
PNAS,
January 21, 2003;
100(2):
645 - 650.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
W. Piacibello, S. Bruno, F. Sanavio, S. Droetto, M. Gunetti, L. Ailles, F. S. de Sio, A. Viale, L. Gammaitoni, A. Lombardo, et al.
Lentiviral gene transfer and ex vivo expansion of human primitive stem cells capable of primary, secondary, and tertiary multilineage repopulation in NOD/SCID mice
Blood,
December 15, 2002;
100(13):
4391 - 4400.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. P. Chute, A. A. Saini, D. J. Chute, M. R. Wells, W. B. Clark, D. M. Harlan, J. Park, M. K. Stull, C. Civin, and T. A. Davis
Ex vivo culture with human brain endothelial cells increases the SCID-repopulating capacity of adult human bone marrow
Blood,
December 15, 2002;
100(13):
4433 - 4439.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Challier, L. Cocault, R. Berthier, N. Binart, I. Dusanter-Fourt, G. Uzan, and M. Souyri
The cytoplasmic domain of Mpl receptor transduces exclusive signals in embryonic and fetal hematopoietic cells
Blood,
August 28, 2002;
100(6):
2063 - 2070.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
N. C. Josephson, G. Vassilopoulos, G. D. Trobridge, G. V. Priestley, B. L. Wood, T. Papayannopoulou, and D. W. Russell
Transduction of human NOD/SCID-repopulating cells with both lymphoid and myeloid potential by foamy virus vectors
PNAS,
June 11, 2002;
99(12):
8295 - 8300.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. Glimm, P. Tang, I. Clark-Lewis, C. von Kalle, and C. Eaves
Ex vivo treatment of proliferating human cord blood stem cells with stroma-derived factor-1 enhances their ability to engraft NOD/SCID mice
Blood,
May 1, 2002;
99(9):
3454 - 3457.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Grande, M. Montanari, E. Tagliafico, R. Manfredini, T. Z. Marani, M. Siena, E. Tenedini, A. Gallinelli, and S. Ferrari
Physiological levels of 1{alpha}, 25 dihydroxyvitamin D3 induce the monocytic commitment of CD34+ hematopoietic progenitors
J. Leukoc. Biol.,
April 1, 2002;
71(4):
641 - 651.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Tonnelle, F. Bardin, C. Maroc, A.-M. Imbert, F. Campa, A. Dalloul, C. Schmitt, and C. Chabannon
Forced expression of the Ikaros 6 isoform in human placental blood CD34+ cells impairs their ability to differentiate toward the B-lymphoid lineage
Blood,
November 1, 2001;
98(9):
2673 - 2680.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
I. Dybedal, D. Bryder, A. Fossum, L. S. Rusten, and S. E. W. Jacobsen
Tumor necrosis factor (TNF)-mediated activation of the p55 TNF receptor negatively regulates maintenance of cycling reconstituting human hematopoietic stem cells
Blood,
September 15, 2001;
98(6):
1782 - 1791.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
I. D. Lewis, G. Almeida-Porada, J. Du, I. R. Lemischka, K. A. Moore, E. D. Zanjani, and C. M. Verfaillie
Umbilical cord blood cells capable of engrafting in primary, secondary, and tertiary xenogeneic hosts are preserved after ex vivo culture in a noncontact system
Blood,
June 1, 2001;
97(11):
3441 - 3449.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. E. Perez, H. M. Rinder, C. Wang, J. B. Tracey, N. Maun, and D. S. Krause
Xenotransplantation of immunodeficient mice with mobilized human blood CD34+ cells provides an in vivo model for human megakaryocytopoiesis and platelet production
Blood,
March 15, 2001;
97(6):
1635 - 1643.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. Glimm, I.-H. Oh, and C. J. Eaves
Human hematopoietic stem cells stimulated to proliferate in vitro lose engraftment potential during their S/G2/M transit and do not reenter G0
Blood,
December 15, 2000;
96(13):
4185 - 4193.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. Salmon, V. Kindler, O. Ducrey, B. Chapuis, R. H. Zubler, and D. Trono
High-level transgene expression in human hematopoietic progenitors and differentiated blood lineages after transduction with improved lentiviral vectors
Blood,
November 15, 2000;
96(10):
3392 - 3398.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. S. Rosler, J. E. Brandt, J. Chute, and R. Hoffman
An in vivo competitive repopulation assay for various sources of human hematopoietic stem cells
Blood,
November 15, 2000;
96(10):
3414 - 3421.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. Hennemann, I.-H. Oh, J. Y. Chuo, C. P. Kalberer, P. D. Schley, S. Rose-John, R. K. Humphries, and C. J. Eaves
Efficient retrovirus-mediated gene transfer to transplantable human bone marrow cells in the absence of fibronectin
Blood,
October 1, 2000;
96(7):
2432 - 2439.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. Bryder and S. E. W. Jacobsen
Interleukin-3 supports expansion of long-term multilineage repopulating activity after multiple stem cell divisions in vitro
Blood,
September 1, 2000;
96(5):
1748 - 1755.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. W. Zandstra, D. A. Lauffenburger, and C. J. Eaves
A ligand-receptor signaling threshold model of stem cell differentiation control: a biologically conserved mechanism applicable to hematopoiesis
Blood,
August 15, 2000;
96(4):
1215 - 1222.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
O. Kollet, A. Peled, T. Byk, H. Ben-Hur, D. Greiner, L. Shultz, and T. Lapidot
beta 2 Microglobulin-deficient (B2mnull) NOD/SCID mice are excellent recipients for studying human stem cell function
Blood,
May 15, 2000;
95(10):
3102 - 3105.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. Verhasselt, T. Kerre, E. Naessens, D. Vanhecke, M. De Smedt, B. Vandekerckhove, and J. Plum
Thymic Repopulation by CD34+ Human Cord Blood Cells After Expansion in Stroma-Free Culture
Blood,
December 1, 1999;
94(11):
3644 - 3652.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
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TOP
ABSTRACT
INTRODUCTION