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1999 93: 3736-3749
Fagioli, Eliana Perissinotto, Giuliana Cavalloni, Orit Kollet, Tsvee Lapidot and Massimo Aglietta
Wanda Piacibello, Fiorella Sanavio, Antonella Severino, Alessandra Danè, Loretta Gammaitoni, Franca
the Amplification and Self-Renewal of Repopulating Stem Cells
of Human CD34+ Cord Blood Cells After Ex Vivo Expansion: Evidence for
Engraftment in Nonobese Diabetic Severe Combined Immunodeficient Mice
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Copyright 2011 by The American Society of Hematology; all rights reserved.
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Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by
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HEMATOPOIESIS
Engraftment in Nonobese Diabetic Severe Combined Immunodeficient
Mice of Human CD34ⴙCord 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 Dane`, Loretta Gammaitoni,
Franca Fagioli, Eliana Perissinotto, Giuliana Cavalloni, Orit Kollet, Tsvee Lapidot, and Massimo Aglietta
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 CD34ⴙcord 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 determin-
ing this phenomenon. However, the effect of these manipula-
tions 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 hematopoi-
etic precursors by transplanting human hematopoietic cells
into sublethally irradiated nonobese diabetic severe com-
bined immunodeficient (NOD/SCID) mice. We have exam-
ined here the expansion of cells, CD34ⴙand CD34ⴙ38ⴚ
subpopulations, colony-forming cells (CFC), long-term cul-
ture initiating cells (LTC-IC) and the maintenance or the
expansion of SCID-repopulating cells (SRC) during stroma-
free suspension cultures of human CD34ⴙCB cells for up to
12 weeks. Groups of sublethally irradiated NOD/SCID mice
were injected with either 35,000, 20,000, and 10,000 unma-
nipulated CD34ⴙCB cells, which were cryopreserved at the
start of cultures, or the cryopreserved cells expanded from
35,000, 20,000, or 10,000 CD34ⴙcells for 4, 8, and 12 weeks in
the presence of a combination of early acting recombinant
growthfactors (flt 3/flk2ligand[FL] ⴙmegakaryocyte growth
and development factor [MGDF] ⴞstem cell factor [SCF] ⴞ
interleukin-6[IL-6]). Mice thathadbeen injected with H20,000
fresh or cryopreserved uncultured CD34ⴙcells 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 CD34ⴙCB 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 CD34ⴚcells) suggested that primitive hematopoi-
etic cells were maintained for up to 10 weeks of cultures. In
addition, dilution experiments suggest that SRC are ex-
panded more than 70-fold after 9 to 10 weeks of expansion.
These results support and extend our previous findings that
CD34ⴙCB 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.
r
1999 by The American Society of Hematology.
AN ESSENTIAL PROPERTY of hematopoietic stem cells
is their ability to divide without significant alteration of
their proliferative potential or differentiation state. Characteriza-
tion 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.7Recently, 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 differ-
ent 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 dia-
betic/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
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.
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’Universita`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.
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@mail.ircc.unito.it.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked ‘‘adver-
tisement’’ in accordance with 18 U.S.C. section 1734 solely to indicate
this fact.
r1999 by The American Society of Hematology.
0006-4971/99/9311-0001$3.00/0
3736
Blood,
Vol 93, No 11 (June 1), 1999: pp 3736-3749
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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 progeni-
tor 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 expan-
sion 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. Experi-
ments 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 condi-
tions.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 cocul-
tures 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, megakaryo-
cyte 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.
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 dex-
trose.
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 puri-
fied 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 recombi-
nant human interleukin-3 (rhIL-3) were from Sandoz (Basel, Switzer-
land); 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
EXPANSION OF IN VIVO REPOPULATING STEM CELLS 3737
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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
to2⫻106cells/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 ⱖ99.9% 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 ⫻103CD34⫹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% methylcel-
lulose (Fluka) and IMDM containing 30% FCS at 37°C in a humidified
atmosphere at 5% CO2in 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 progeni-
tors.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 107fresh 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, 10⫺6mol/L hydrocorti-
sone, and penicillin/streptomycin] and by plating the irradiated [15 Gy]
and trypsinized stroma at 7 ⫻103/cm2in 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) Atotal 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 ⫻104CD34⫹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 immunophe-
notype 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
3738 PIACIBELLO ET AL
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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
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 ⫻105and 1 ⫻105CD34⫹cells
Fig 1. Representative DNA analysis of human cell engraftment in
the BM of NOD/SCID mice transplanted with 1 ⴛ104,2ⴛ104, and
3.5 ⴛ104unmanipulated CD34ⴙCB cells. Cells were injected together
with preirradiated 2 ⴛ105CD34ⴚCB 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.
Table 1. Main Characteristics of Injected CB CD34ⴙCells and Their Ability to Engraft the BM of NOD/SCID Recipients
Injected/Mouse No. of
Positive Mice Engraftment (%) Human CFC (⫻103)*CD34⫹Cells CFC LTC-IC
2⫻10549,330 17,800 3/3† 12.5, 21, 7‡ 51.2, 98.4, 27.6
1⫻10524,665 8,900 4/4 1.3, 3, 11, 25 17.7, 14.2, 41.5, 77.6
5⫻10412,332 4,450 4/5 1.4, 16, 3, 8, 0 ND, 74.9, 10.2, 36.4, 0
3.5 ⫻1048,631 3,115 4/6 1.2, 2.3, 0, 0, 1.6, 3 4.9, 9.8, 0, 0, 8.08, 9.5
2⫻1044,933 1,780 3/7 0.18, 0.5, 0.3, 0, 0, 0, 0 0.76, 2.1, 1.9, 0, 0, 0, 0
1⫻1042,466 890 0/5 0, 0, 0, 0, 0 0, 0, 0, 0, 0
CD34⫹cells deriving from 6 different CB samples were separated as described, pooled, and then stored frozen. At the time of injection, the
samples were thawed, washed, counted, resuspended in 300 µL of IMDM containing 10% FCS and then injected into 3 to 7 animals as described.
BM of NOD/SCID recipients was recovered from both the femurs and the tibias at week 8 posttransplant.
Abbreviation: ND, not done.
*A total of 1 ⫻105to5⫻105BM cells of the transplanted mice were seeded in triplicate dishes for plasma clot assays. Recombinant human IL-3,
GM-CSF, SCF, and EPO were added at start of cultures. Dishes were scored after 14 days of incubation, after staining with MoAb as described in
Materials and Methods. Only fluorescent colonies were considered of human origin. Values are calculated taking into account that the BM of both
femurs and tibias represent 25% of the total BM.
†Positive mouse ⫽⬎0.1% of human CD45⫹, CD71⫹, and ␣-GpA A⫹cells in the whole BM on FACSscan analysis.
‡Percent of human CD45⫹, CD71⫹, and GpA⫹cells in the whole BM on FACSscan analysis.
EXPANSION OF IN VIVO REPOPULATING STEM CELLS 3739
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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 CD34ⴙCB cells. (a) Negative control: a nonengrafted mouse (transplanted with 2 ⴛ105irradiated CD34ⴚCB
carrier cells). (Middle) A mouse transplanted with 1 ⴛ105CD34ⴙCB cells. (Bottom) A mouse transplanted with 2 ⴛ104CD34ⴙcells. CD45/CD34
and CD45/CD19 analysis was performed on total BM cells. (B) Multilineage engraftment in the BM of a representative mouse transplanted with
1ⴛ105unmanipulated CD34ⴙCB 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.
Table 2. Comparison Between Three Different Expansion Protocols
Growth
Factors
Input ⫹4wk ⫹6wk ⫹8wk
Demide-
population
Well Exp. Wells Flasks
Demide-
population
Well Exp. Wells Flasks
Demide-
population
Well Exp. Wells Flasks
FL ⫹MGDF
Cells ⫻1060.02 3.7 ⫾1.09 2.97 ⫾0.87 4.8 ⫾1.8 15.2 ⫾7 14.2 ⫾6.4 14.02 ⫾7.9 42.4 ⫾24.4 39.8 ⫾25.6 37.4 ⫾19.9
CD34⫹%91⫾8.5 4.08 ⫾1.0 3.76 ⫾0.98 3.6 ⫾0.99 1.83 ⫾0.6 1.90 ⫾0.8 2.57 ⫾1.1 2.14 ⫾0.9 2.35 ⫾0.87 2.61 ⫾0.77
CFC ⫻1032.8 ⫾1.4 115.2 ⫾48.8 85.2 ⫾41.6 144.1 ⫾77.9 196.7 ⫾135.4 148.9 ⫾110.6 213 ⫾106.7 1,008 ⫾69.5 943.6 ⫾372.2 863.3 ⫾295.8
LTC-IC ⫻1033.6 ⫾0.9 27.5 ⫾17.5 20.6 ⫾11.2 24.7 ⫾11.5 113.2 ⫾14.8 126.8 ⫾36.1 131.6 ⫾51.2 929.6 ⫾659.5 873.2 ⫾312 899.6 ⫾236
SCF ⫹FL ⫹
MGDF
Cells ⫻1060.02 9.09 ⫾3.2 8.89 ⫾2.7 10.3 ⫾1.9 31.28 ⫾8 29.6 ⫾9.6 27.9 ⫾6.9 103.7 ⫾32.9 91 ⫾36.7 106 ⫾40.6
CD34⫹%91⫾8.5 3.28 ⫾0.29 3.61 ⫾1.03 3.02 ⫾0.7 1.67 ⫾0.9 1.58 ⫾0.12 1.7 ⫾0.9 2.97 ⫾0.7 2.06 ⫾1.3 2.06 ⫾0.85
CFC ⫻1032.8 ⫾1.4 194.2 ⫾60.2 177.7 ⫾55.7 255.3 ⫾41 314.7 ⫾99.1 527.1 ⫾85.8 298.7 ⫾106.2 1,252.6 ⫾508.1 1,196.6 ⫾676.4 1,066.5 ⫾737.4
LTC-IC ⫻1033.6 ⫾0.9 48.2 ⫾24.8 46.4 ⫾19.9 57.4 ⫾22 172.8 ⫾122.4 232.4 ⫾150.5 204.6 ⫾79 1,012.9 ⫾795.6 985 ⫾345 1,003 ⫾604
IL6 ⫹SCF ⫹
FL ⫹MGDF
Cells ⫻1060.02 10.01 ⫾3.2 11.74 ⫾2.7 6.96 ⫾2.91 55.6 ⫾21.7 49.5 ⫾19.6 57.1 ⫾23.6 113.4 ⫾49.8 100.4 ⫾38 249 ⫾73.1
CD34⫹%91⫾8.5 4.32 ⫾0.95 3.77 ⫾1.4 3.73 ⫾0.7 2.47 ⫾0.8 1.98 ⫾0.6 2.55 ⫾0.8 2.87 ⫾0.85 1.94 ⫾0.7 2.03 ⫾0.9
CFC ⫻1032.8 ⫾1.4 180.6 ⫾46.8 153.5 ⫾57.1 223.2 ⫾58.8 473 ⫾16 416.2 ⫾91 389.9 ⫾76.1 1,898.6 ⫾1,785 1,679.9 2,696 ⫾863.4
LTC-IC ⫻1033.6 ⫾0.9 52.2 ⫾21.9 41.2 ⫾23.4 55.2 ⫾41 290.6 ⫾78.5 215.7 ⫾48 256.4 ⫾91.3 1,100.5 ⫾665 903.6 ⫾114.1 1,264 ⫾592
Twenty thousand CB CD34⫹cells were resuspended in 1 mL IMDM containing 10% pooled human serum and cultured for up to 8 weeks in 24-well plates. In the first
series(demidepopulationwells)cellsin1mLofasinglewellweredemidepopulatedeveryweekbyremovalofone half the culture medium (and cells), which was replaced
with the same volume of fresh medium and growth factors. In the second series (expansion wells), 2 ⫻104CD34⫹cells were prepared as described above in 24-well plates
in 1 mL. Every week the cells were resuspended in twice the volume of culture medium, so that every week additional new 24-wells were set up. In the third series (flasks)
2⫻104CD34⫹cells/mL were layered on the bottom of T flasks and every week the culture volume was doubled. At the indicated time points, the cells were harvested,
counted, and aliquots analyzed for CD34⫹antigen expression. Suitable aliquots were assayed in semisolid assays for CFC and on stroma cocultures for LTC-1C
enumeration. Mean ⫾SEM of 4 to 6 separate experiments performed in quadruplicate.
3740 PIACIBELLO ET AL
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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 engraft-
ment 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).
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
Table 3. Antigenic Composition of CD34ⴙCB Cells at Initiation of Liquid Cultures and at Various Time Intervals During Ex Vivo Expansion
Culture
Conditions CD34 CD38 CD34⫹38⫺CD33 CD13 CD33⫹13⫹CD14 CD2 CD19 CD41 ␣GpA CD71
Start 91 ⫾8.5 94 ⫾3.1 0.95 ⫾0.05 49 ⫾6.4 0 0 0.3 ⫾0.03 2.0 ⫾1.6 1.8 ⫾1.1 0.4 ⫾0.2 4.1 ⫾1.6 0
wk 4 3.9 ⫾0.4 76 ⫾4.8 0.18 ⫾0.04 96 ⫾13 94.7 ⫾7.8 91 ⫾11.6 59 ⫾7.1 0.25 ⫾0.04 0.9 ⫾0.2 3 ⫾1.4 5 ⫾22⫾2
wk 8 2.1 ⫾0.17 27 ⫾70.6⫾0.15 32 ⫾9.6 1.72 ⫾16.4 1.7 ⫾0.3 25 ⫾1.4 0.12 ⫾0.01 0.07 ⫾0.01 0.5 ⫾0.01 0.3 ⫾0.01 2.63 ⫾1.6
wk 12 2.3 ⫾134⫾11 0.46 ⫾0.9 85.4 ⫾2.4 7.2 ⫾4.3 7.1 ⫾2.7 36.3 ⫾2.1 0.1 ⫾0 0.19 ⫾0.03 0.18 ⫾0.6 0.56 ⫾0.04 13.1 ⫾2.9
Positive cells (%) of each subpopulation. Mean ⫾standard deviation (SD) from four to seven separate experiments performed in replicate wells for each growth factor
combination at different time points.
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
CB Cells Growth Factors Added
Injected/Mouse Engraftment
Cells ⫻106CFC ⫻103LTC-IC ⫻103% Human
Cells† Human CFC/Mouse
⫻103‡
EXP. 1 Start of cultures 0.02 2.2 ⫾0.7 4.62 ⫾0.36 0.18, 0 0.86, 0
4 weeks of cultures
FL ⫹MGDF 9.9 ⫾2.8 (495)* 116.2 ⫾18.6 (52.8)* 23.42 ⫾3.3 (5.06)* 10.4, 12 20.48 ⫾0.4, 86.74 ⫾1.3
SCF ⫹FL ⫹MGDF 11.7 ⫾3.1 (585)* 79.3 ⫾10.2 (36)* 29.97 ⫾5.1 (6.48)* 11, 15 ND, 54.71 ⫾2.7
IL6 ⫹SCF ⫹FL ⫹MGDF 7.4 ⫾1.2 (370)* 111.6 ⫾17.8 (50.7)* 23.58 ⫾4.2 (5.12)* 21, 6 25.7 ⫾4.6, 46.72 ⫾3.7
EXP. 2 Start of cultures 0.02 2.46 ⫾0.6 2.55 ⫾0.4 0, 0.3 0, 1.68
4 weeks of cultures
FL ⫹MGDF 4.22 ⫾0.77 (211)* 48.48 ⫾7.1 (19.7)* 26.55 ⫾4.7 (11.8)* 9.8, 0 74.26 ⫾6.7, 0
SCF ⫹FL ⫹MGDF 5.38 ⫾0.98 (269)* 59.89 ⫾8.9 (24.3)* 25.82 ⫾6.1 (11.5)* ND, 21 ND, 59.64 ⫾6.4
IL6 ⫹SCF ⫹FL ⫹MGDF 5.6 ⫾1.2 (280)* 70.72 ⫾10.6 (28.7)* 30.41 ⫾4.6 (13.5)* 22.8, 16 ND, 114.71 ⫾11.6
EXP. 3 Start of cultures 0.01 0.52 ⫾0.5 1.7 ⫾0.6 0, 0 0, 0
4 weeks of cultures
FL ⫹MGDF 4.16 ⫾0.89 (416)* 34.24 ⫾7.9 (68.5)* 38.65 ⫾5.1 (22.7)* 10.5, 13 ND, 39.42 ⫾5.2
SCF ⫹FL ⫹MGDF 6.9 ⫾1.5 (690)* 43.65 ⫾6.9 (83.9)* ND — 16, 12 43.74 ⫾5, 110.71 ⫾12.6
IL6 ⫹SCF ⫹FL ⫹MGDF 7.2 ⫾2.2 (720)* 42.49 ⫾6.3 (81.7)* 41.69 ⫾8.8 (24.5)* 14, 0 165.71 ⫾13, 0
CD34⫹cells were cultured at 10 ⫻103/mLor20⫻103/mL in 24-well plates as described. Cells at the start of cultures and cells harvested after 4
weeks of suspension cultures were cryopreserved and kept frozen until the time of injection in NOD/SCID mice. The expansion cultures were
performed in quadruplicate. Two mice were injected each with 20,000 or 10,000 uncultured cells and two mice each with the content of a single
expansion culture.
*Fold increase (compared with the input value).
†Each value represents the percentage of human CD45⫹, CD71⫹, and ␣-GpA⫹cells detected in the BM of each individual mouse.
‡Mean ⫾SEM of the number of human CFU-GM ⫹BFU-E ⫹CFU-GEMM ⫹CFU-Mk generated by plating 1 ⫻105to5⫻105BM cells of engrafted
NOD/SCID mice (at least three dishes per point), taking into account that BM cells of femurs and tibias represent 25% of the total BM.
EXPANSION OF IN VIVO REPOPULATING STEM CELLS 3741
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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).
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.
In keeping with the results reported in Table 1, only 2 of 6
mice that had been injected with 20,000 or 10,000 cryopre-
served 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 anti-
bodies. All of the colonies were positive, thus indicating their
human origin.
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
Fig 3. Engraftment of human CB CD34ⴙcells 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 determin-
ing the percent of human CD45ⴙ, CD71ⴙ, and GpAⴙcells within the
total BM cells in individual NOD/SCID mice. Each animal was injected
either with unmanipulated CD34ⴙCB cells (2 ⴛ104,䊉;3.5ⴛ104,䊊)
either with the cells generated by initial 2 ⴛ104or 3.5 ⴛ104CD34ⴙ
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.
Fig 4. Representative Southern blot analysis of individual NOD/
SCID mice transplanted with 1 ⴛ104,2ⴛ104, and 3.5 ⴛ104
unmanipulated CD34ⴙCB cells and with week 4 expanded cells
(deriving from 1 ⴛ104initial CD34ⴙcells). DNA was extracted from
the murine BM at week 8 after transplant and hybridized with a
human chromosome 17–specific ␣-satellite probe.
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proportion of the total cell population ( Fig 5). CD34⫹CD38⫺
cells were also detected (Fig 6).
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
Fig 5. Analysis of CD34 anti-
gen expression on CB CD34ⴙ
cells at start of cultures (week 0)
and at weeks 4, 6, 8, and 12 of ex
vivo expansion in stroma-free
culturessupplemented with FLⴙ
MGDF and IL6ⴙSCFⴙFLⴙMGDF.
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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.
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 hemato-
poietic 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 com-
pletely engraft the BM of sublethally irradiated NOD/SCID
Fig 6. Analysis of CD34 and
CD38 antigen expression on
CD34ⴙCB cells at start of cultures
(week 0) and after 4 and 8 weeks
of expansion in stroma-free cul-
tures supplemented with FLⴙ
MGDF and IL6ⴙSCFⴙFLⴙMGDF.
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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, commit-
ted unipotent and multipotent progenitors occurred; also primi-
tive 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 expan-
sion 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 demidepopula-
tion assay could be reproduced in a larger scale expansion
Fig 7. (A) Representative FACS profiles of marrow cells from individual NOD/SCID mice transplanted 8 weeks previously with cells deriving
from 3.5 ⴛ104CD34ⴙCB 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 ⴛ104initial CD34ⴙcells. 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 ⴛ104initial CD34ⴙcells). 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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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.
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 repre-
sents a mouse) were injected with fractions of the expanded cells,
corresponding to numbers of initial CD34ⴙcells 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.
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
In Culture Injected/Mouse Engraftment
Source
of Cells Growth Factors
Added Cells
(fold increase) CFC
(fold increase) LTC-IC
(fold increase) Cells CFC LTC-IC % of Human
Cells* Human CFC/
mouse ⫻103†
Start 10,000 866.4 1,332 10,000 866.4 1,332 0, 0 0, 0
5,000 433 666 0, 0 0, 0
2,500 216 333 0, 0 0, 0
1,250 108 166 0, 0 0, 0
4 weeks ⫹IL-6 ⫹SCF ⫹
FL ⫹MGDF 3.16 ⫻106
(316) 37,184
(43) 11,248
(8.4) 3.160 ⫻106
0.395 ⫻10637,184
4,648 11,248
1,456 8.5, 9.9
2.5, 3 15.64, 18.4
7.2, 9.6
8 weeks ⫹IL-6 ⫹SCF ⫹
FL ⫹MGDF 28.99 ⫻106
(2,899) 129,464
(149) 56,304
(42) 28.99 ⫻106
3.60 ⫻106129,464
16,183 56,304
7,038 7.3, 18
2.8, 3.6 18.22, 36.72
ND, 34.32
1.80 ⫻1068,091 3,519 0.7, 1.9 1.34, 3.87
12 weeks ⫹IL-6 ⫹SCF ⫹
FL ⫹MGDF 109 ⫻106
(10,900) 2,909,061
(3,358) 382,284
(287) 109 ⫻106
13.6 ⫻106—
363,632 —
47,785 ——
11, 16 34.98, 50.76
6.8 ⫻106181,816 23,892 8, 6.8 23.44, 44.66
3.4 ⫻10690,908 11,946 1.4, 0.9 12.6, 19.3
Start 20,000 2,248 3,632 20,000 3,248 3,632 0, 0 2.08, 0
5,000 812 908 0, 0 0, 0
2,500 406 454 0, 0 0, 0
1,250 203 227 0, 0 0, 0
7 weeks FL ⫹MGDF 22 ⫻106
(1,100) 372,736
(166) 90,112
(25) 22 ⫻106
2.75 ⫻106124,930
15,616 90,112
11,264 6.8, 15
4, 11 29.9, 63.84
8.3, 75.9
1.37 ⫻1067,808 5,632 0, 2.6 0, 13.4
SCF ⫹FL ⫹
MGDF 36.4 ⫻106
(1,820) 381,728
(170) 126,976
(35) 36.40 ⫻106
4.55 ⫻106381,728
47,716 126,976
15,872 7.8, 14.2
5.6, 9 41.87, 46.86
2.08, ND
2.27 ⫻10623,858 7,936 0.9, 3 2.54, 10.1
IL-6 ⫹SCF ⫹
FL ⫹MGDF 17.8 ⫻106
(890) 361,081
(160) 102,400
(28) 17.80 ⫻106
2.22 ⫻106361,081
45,135 102,400
12,800 18, 10.4
4, 6 108.9, 54.16
ND, 23.9
1.11 ⫻10622,567 6,400 1, 2.1 6.32, 15.6
12 weeks IL-6 ⫹SCF ⫹
FL ⫹MGDF 710 ⫻106
(35,500) 2,928,640
(1,303) 980,460
(270) —
88.75 ⫻106—
366,080 —
122,580 ——
ND, ND ——
ND, ND
22.20 ⫻10691,520 30,641 11, 6.5 50.16, 30.07
11.10 ⫻10645,760 15,322 0.6, 3.9 1.87, 6
5.50 ⫻10622,880 7,661 0.9, 0.5 5.56, 2.3
The value in parentheses represents the fold increase compared with input values.
*Each value represents the percent of human CD45⫹, CD71⫹, and ␣-GPA⫹cells in the BM of each individual mouse.
†As explained in Table 4. Expansion studies were performed in quadruplicate. Two mice were injected with each cell dose as indicated.
3746 PIACIBELLO ET AL
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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 distribu-
tion between the two CB subsets as LTC-IC. It was concluded
that conditions, which were able to stimulate LTC-IC expan-
sion, 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 condi-
tions; 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 recipi-
ents; 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 increas-
ingly 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 hemato-
poietic 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 imma-
ture hematopoietic cells and for various experimental purposes,
such as required for the development of efficient gene transfer
protocols into hematopoietic cells with retention of repopulat-
ing ability.
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