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Blood, Vol. 95 No. 1 (January 1), 2000:
pp. 102-110
HEMATOPOIESIS
Expansion of human cord blood CD34+CD38
cells in ex vivo culture during retroviral transduction without a
corresponding increase in SCID repopulating cell (SRC) frequency:
dissociation of SRC phenotype and function
Craig Dorrell,
Olga I. Gan,
Daniel S. Pereira,
Robert G. Hawley, and
John E. Dick
From Programs in Cancer/Blood and Developmental Biology, Hospital
for Sick Children; Department of Molecular and Medical Genetics,
University of Toronto; and The Toronto Hospital, Toronto, Ontario,
Canada.
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Abstract |
Current procedures for the genetic manipulation of hematopoietic
stem cells are relatively inefficient due, in part, to a poor
understanding of the conditions for ex vivo maintenance or expansion of
stem cells. We report improvements in the retroviral transduction of
human stem cells based on the SCID-repopulating cell (SRC) assay and
analysis of Lin CD34+CD38
cells as a surrogate measure of stem cell function. Based on our
earlier study of the conditions required for ex vivo expansion of
LinCD34+ CD38 cells and
SRC, CD34+-enriched lineage-depleted umbilical cord
blood cells were cultured for 2 to 6 days on fibronectin fragment in
MGIN (MSCV-EGFP-Neo) retroviral supernatant (containing 1.5% fetal
bovine serum) and IL-6, SCF, Flt-3 ligand, and G-CSF. Both
CD34+CD38 cells (20.8%) and CFC (26.3%)
were efficiently marked. When the bone marrow of engrafted NOD/SCID
mice was examined, 75% (12/16) contained multilineage (myeloid and B
lymphoid) EGFP+ human cells composing as much as 59% of
the graft. Half of these mice received a limiting dose of SRC,
suggesting that the marked cells were derived from a single transduced
SRC. Surprisingly, these culture conditions produced a large
expansion (166-fold) of cells with the
CD34+CD38 phenotype (n = 20). However,
there was no increase in SRC numbers, indicating dissociation between
the CD34+CD38 phenotype and SRC function.
The underlying mechanism involved apparent downregulation of CD38
expression within a population of cultured
CD34+CD38+ cells that no longer contained
any SRC function. These results suggest that the relationship between
stem cell function and cell surface phenotype may not be reliable for
cultured cells. (Blood. 2000;95:102-110)
© 2000 by The American Society of Hematology.
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Introduction |
The expansion of human hematopoietic repopulating cells
in ex vivo culture will likely have important applications in
transplantation, stem cell marking, and gene therapy. Expansion may
allow the use of stem cell sources, which are available in limited
quantities, such as umbilical cord blood (CB), and should permit the
retroviral transduction of stem cells for the long-term correction of
genetic defects.1 Extensive amplification of primitive
progenitor cells assayed as colony-forming cells (CFC) or cobblestone
area-forming cells (CAFC) / long-term culture-initiating cells (LTC-IC)
in vitro2-5 has been achieved, but the repopulating
activity of cells produced in these cultures was not tested. While the
relationship between CAFC/LTC-IC and the stem cell remains unclear,
experiments using the SCID repopulating cell (SRC) stem cell
model6,7 have demonstrated that several properties
distinguish SRC from LTC-IC and suggest that the former is a useful
predictor of stem cell activity.8-10 Recently, this
repopulation model has been applied to cells grown under ex vivo
expansion conditions, revealing that SRC numbers can be increased 2- to
4-fold in 4-day serum-free suspension culture.11,12
Culture conditions that induce stem cell cycling without causing
differentiation are necessary for gene transfer using Moloney murine
leukemia virus-based retroviral vectors, because mitotic activity is
required for proviral integration.13 Early attempts to
retrovirally transduce SRC were inefficient; under conditions that
permitted the relatively high marking of CFC and LTC-IC, SRC were
poorly infected.8 This result parallels that of human gene
therapy trials using similar retroviral vectors, which did not achieve
significant levels of gene transfer in transplanted recipients,14-18 and suggests that the SRC model is a
useful predictor of clinical stem cell behavior. Recent reports have
used the SRC assay to improve the efficiency of transduction using new
vectors and ex vivo culture protocols.19-23 Although the
conditions differ among the reports, most mice now contain marked human
cells with transduction frequencies of approximately 20%. However, the
growth and differentiation behavior of SRC in these culture conditions is unclear19-22 and, in general, large numbers of cells
needed to be transplanted, suggesting that SRC may be lost during the transduction. Rebel et al23 were the first to examine this
question in detail, and they found a significant decline of
approximately 4-fold both in the frequency and total number of SRC
under their culture conditions.
With the advent of retroviral vectors expressing selectable markers
such as enhanced green fluorescent protein (EGFP), it will be possible
to rapidly optimize expansion/transduction protocols to favor the
efficient transduction of primitive hematopoietic cell subsets,
assuming that there is a clear understanding of the cell surface
phenotype that correlates with stem cell function. It is generally
accepted that CD34 expression correlates inversely with hematopoietic
differentiation.24 The membrane-bound ectoenzyme CD38 has
also been used to classify the differentiation state of hematopoietic
cells. Among lineage-depleted (Lin) cells, a
CD34+CD38 phenotype identifies a very rare
and highly primitive hematopoietic subpopulation in both fetal tissues
and adult bone marrow.25-28 The CB
LinCD34+CD38 subpopulation
contains SRC at a frequency of 1 in 617, while LinCD34+CD38+ cells lack SRC
function.9 Furthermore, Bhatia et al11 showed that virtually all new cells produced within 4 days in SRC expansion cultures initiated with CD34+CD38 cells
retained this phenotype, indicating that newly generated SRC are
CD34+CD38. In contrast, the
CD34+CD38+ cells generated following longer
times of culture (8 days) no longer had SRC activity, providing further
evidence of a link between stem cell function and cell surface
phenotype. The CD34+CD38 surface phenotype
has also been used as a direct measure of primitive cell expansion in
culture.4 Together, these publications suggest that the
differentiation state of infected cells can be conveniently monitored
by simultaneous measurement of CD34 and CD38 expression and gene
transfer using EGFP.
In this study, we developed an ex vivo infection protocol that combines
retroviral transduction of Lin cells with ex vivo
expansion culture conditions adapted from those previously reported by
Bhatia et al11 and Conneally et al.12 SRC
transduction was significantly improved compared with our earlier
study, consistent with recent reports. Unlike other quantitative
studies that have documented a loss of SRC,23 the ex vivo
transduction conditions we used resulted in the maintenance of SRC
number over 4 days of culture. In addition, these studies also showed a
clear dissociation between the CD34+CD38
cell surface phenotype and SRC function, indicating that this combination of cell surface markers may not be reliable with cultured cells.
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Materials and methods |
Sample collection and purification
CB samples were obtained from placental and umbilical tissues
scheduled for discard according to procedures approved by the institutional review board of Mount Sinai Hospital (Toronto, Canada). Samples were collected in heparin and centrifuged on Ficoll-Paque (Pharmacia, Uppsala, Sweden) to obtain mononuclear cells. Lineage depletion and CD34+-enrichment were achieved by negative
selection with the StemSepTM system according to the manufacturer's
protocol (Stem Cell Technologies Inc, Vancouver, British Columbia,
Canada). The antibody cocktail that was used removes cells expressing
glycophorin A, CD2, CD3, CD14, CD16, CD19, CD24, CD41, CD56, or CD66b.
The efficiency of primitive cell enrichment was determined by flow
cytometric assessment of CD34 expression (see below).
Flow cytometry
Flow cytometric analysis was performed using a FACScaliburTM or
FACStarTM Plus (Becton Dickinson, San Jose, CA).
Isotype controls were mouse immunoglobulin G conjugated to fluorescein
isothiocyanate (FITC), phycoerythrin (PE), peridinin chlorophyll
protein (PerCP; all Becton Dickinson), or tricolour (TC; Caltag,
Burlingame, CA). The CD34 and CD38 expression characteristics of
preculture and postculture CB cells were assessed using anti-CD34-PerCP
with anti-CD38-PE (both Becton Dickinson), anti-CD34-PE (Becton
Dickinson) with anti-CD38-TC (Caltag), or anti-CD34-PerCP (Becton
Dickinson) with anti-CD38-FITC (Coulter, Fullerton, CA).
Further characterization was performed using anti-HLADR-PE (Becton
Dickinson), anti-Thy-1-PE (Pharmingen), or anti-CD13-PE
(both Coulter). Marrow cells from transplanted nonobese diabetic/severe
combined immunodeficient (NOD/SCID) mice were assessed using
anti-CD45-PerCP, anti-CD19-PE, and anti-CD33-PE (all Becton Dickinson).
EGFP fluorescence was detected using detector channel FL1 calibrated to
the FITC emission profile. During quadrant analysis, coordinates were
set to locate  99% of isotype events in the lower left quadrant.
Retrovirus production
MGIN is a murine stem cell virus (MSCV)-based retroviral vector in
which EGFP and neomycin phosphotransferase (Neo) are encoded by a
bicistronic transcript expressed from the MSCV long terminal repeat.29 PG13/MGIN producer cells, generated according to
previously published procedures,30 exported gibbon ape
leukemia virus (GALV)-pseudotyped MGIN virus at a titer of
1 × 106 G418-resistant colony-forming units/mL when
assayed on HT1080 human fibrosarcoma cells (CCL-121; American Type
Culture Collection, Manassas, VA). Retroviral supernatants were
collected from subconfluent cultures 12 hours after the medium was
changed to Iscove's modified Dulbecco's medium (IMDM; Gibco BRL,
Burlington, Ontario, Canada) supplemented with 1.5% fetal
bovine serum (FBS; Cansera, Rexdale, Ontario, Canada).
Infection protocol
Infections of LinCB cells were carried out in
flat-bottom 24-well plates or 35-mm dishes (Nunc, Burlington, Ontario,
Canada) that were coated with CH-296 fibronectin fragment
(RetronectinTM; Takara Shuzo Ltd, Otsu, Japan) at 4 µg/cm.2 Cells were deposited at a density of
5 × 104 per well in 1 mL when using 24-well plates
and between 0.3 × 106 to
1.0 × 106 in 2 mL per 35-mm dish. Culture medium
consisted of IMDM supplemented with 1.5% FBS, 20% BIT (bovine serum
albumin, insulin, and human transferrin; Stem Cell Technologies Inc),
100 µM  -mercaptoethanol, and cytokines at 37°C and 5%
CO2. The cytokine mixture included 10 ng/mL interleukin
(IL)-6, 10 ng/mL granulocyte colony-stimulating factor (G-CSF), 300 ng/mL stem cell factor (each provided by Amgen, Thousand Oaks, CA) and
300 ng/mL Flt-3 ligand (Immunex, Seattle, WA). After a 12-hour
prestimulation, the medium was replaced with supernatant from the
PG13/MGIN producer cells and supplemented with BIT,
-mercaptoethanol, and cytokines as described above. The
duration of culture was 48 or 96 hours, and media were replaced with
fresh retroviral supernatant every 12 hours.
Culture of sorted populations to assess postculture CD34/CD38
phenotype
LinCB cells were labeled with anti-CD34-Cy5PE and
anti-CD38-PE and sorted to isolate
CD34CD38,
CD34+CD38, and
CD34+CD38+ fractions using a FACSstarTM Plus.
CD34+CD38 cells were seeded at
1 × 104 cells per well of a 96-well plate (Nunc) in
0.2 mL of culture medium (using the nutrients and growth factors
described above). CD34CD38or
CD34+CD38+ cells were seeded in a 24-well
plate (1 × 105 cells per well in 1.0 mL of medium).
After 4 days at 37°C and 5% CO2, cells were harvested
and characterized for CD34 and CD38 expression by flow cytometry.
Fluorescence microscopy
A Leica inverted fluorescent microscope (Leica,
Heerbrugg, Switzerland) was employed to examine colonies in
methylcellulose (CFC assay) and cobblestone or hematopoietic areas on
stroma (CAFC assay) for EGFP fluorescence.
Progenitor assays
CFC assays were performed as previously described31
except that 10% 5637-conditioned medium was included with uncultured and postculture CB cells. CAFC assays were also performed according to
the previously published protocol.32,33 Briefly,
nonirradiated murine MS-5 cells (generously provided by Kirin Brewery,
Tokyo, Japan) were seeded into 96-well tissue culture plates (Nunc) 1 day prior to plating hematopoietic cells. Uncultured or postculture Lin cells were added at 15 to 100 cells per well in 150 µL of human long-term bone marrow culture media (Stem Cell
Technologies Inc). No hydrocortisone was added to the culture media.
Wells containing at least 1 hematopoietic or cobblestone area were
defined as positive. Positive wells were tested for EGFP fluorescence
by flow cytometric analysis and examination with a fluorescent
microscope. The absolute number of CAFC present in each fraction was
calculated using Poisson statistics.
Analysis of SRC by NOD/SCID mouse repopulation
Primary and cultured cells were transplanted into NOD/SCID mice
using a slightly modified version of our standard
protocol.8 Following transplantation, mice received
intraperitoneal injections of human IL-3 and GM-CSF (6 µg each;
Amgen) on alternating days for the first week posttransplantation. The
combination of brief cytokine treatment and large cell doses
(>105 cells) has been shown to provide optimal
engraftment.34 After 6 to 7 weeks, mice were killed and
bone marrow was collected from femurs and tibiae. Human cell content
was quantified by Southern analysis using a human chromosome
17-specific  -satellite probe.35 Flow cytometic
analysis of the human-specific pan-leukocyte marker CD45 and
human-specific CFC progenitor assays31 were also applied to
each experimental animal. Only mice with 0.1% total human content
and whose marrow contained human CFC were considered to be
engrafted. Gene transfer into human myeloid
(CD45+CD33+) and B lymphoid
(CD45+CD19+) cells was determined by flow
cytometric measurement of EGFP fluorescence; gene transfer into myeloid
progenitors was measured in CFC assays with 1500 µg/mL G418 selection
and verified by observation of EGFP fluorescence.
Statistical analysis
For limiting dilution assays of CAFC and SRC, Poisson statistics for
the single-hit model were applied. The frequency of CAFC and SRC in
cell suspensions was calculated using maximum likelihood estimator.36
| |
Results |
Ex vivo culture increases the number of mononuclear,
CD34+CD38, and progenitor cells
Modifications to the ex vivo culture protocols of Bhatia et
al11 and Conneally et al12 were implemented to
incorporate retroviral gene transfer. Lin cells were
substituted for sorted CD34+CD38 cells
because the former are conveniently obtained and cell sorting reduces
the final yield of primitive cells. We omitted IL-3 because evidence
exists that it can contribute to the induction of differentiation in
early hematopoietic progenitors.37,38 Infection was
facilitated with the application of RetronectinTM to colocalize cells
and virus and with a low (1.5%) concentration of FBS to support
optimal retrovirus production by producer cells. Finally, supernatant was replaced at 12-hour intervals to introduce new retrovirus, which in
turn mandated the addition of fresh growth factors and nutrients.
The differentiation characteristics of Lin CB cells in
these ex vivo infection conditions were first assessed on the basis of
CD34 and CD38 expression. Figure 1 shows
the flow cytometric analysis of a representative 4-day time-course
experiment. The day 0 CD34/CD38 panel depicts the profile of a typical
Lin CB sample immediately following lineage depletion.
CD34+ cells and CD34+CD38 cells
constituted 28% to 78% (mean, 53%; n = 15) and 0.4% to 5.5%
(mean, 1.7%; n = 15) of the total cell number, respectively, defined
by strict gating to exclude nonspecific labeling. It should be noted
that while identical flow cytometer instrument settings were used, an
increase in cellular autofluorescence over the course of the culture
was observed in the isotype analyses. As a result, it was necessary to
adjust quadrant gating to maintain exclusion of >99% of
nonspecifically labeled cells. During days 1 to 4, as illustrated in
Figure 1 and listed in Table 1, the
percentage of CD34+ cells increased slightly. However,
following day 1 a dramatic increase of
CD34+CD38 cell frequency occurred. After 4 days, the CD34+CD38 fraction represented
21% to 48% (mean, 30.6%; n = 14) of the total population. While
the expression of CD34 and CD38 was routinely assayed with
anti-CD34-PerCP and anti-CD38-PE on a FACScaliburTM, the validity of
the observed phenotype was confirmed with alternate antibody
combinations (including anti-CD34-PE with anti-CD38-TC and
anti-CD34-PerCP with anti-CD38-FITC) and by analysis on a FACStarTM
Plus flow sorter (data not shown).
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| Fig 1.
The CD34/CD38 phenotype of Lin CB cells
changes during ex vivo transduction culture.
In this representative experiment (n = 3), uncultured (day 0) or
cultured (days 1 to 4) cells were labeled with PerCP- and PE-conjugated
mouse immunoglobulin G as isotype controls (upper panels) or
anti-CD34-PerCP and anti-CD38-PE (lower panels) and analyzed by flow
cytometry. Quadrants were set to locate at least 99% of the
nonspecific events in the lower left.
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Mean expansion values for total cell count and numbers of
CD34+ and CD34+CD38 cells from
all experiments are given in Table 1. Most striking is the behavior of
the CD34+CD38 fraction, which exhibited a
mean expansion of 166-fold after 4 days. Also shown are the results
obtained from CFC and CAFC assays performed using preculture and
postculture cells. Day 0 CFC frequencies ranged from 11% to 35%
(mean, 26.5%; n = 13), and the day 4 frequencies were moderately
higher, 22% to 36% (mean, 34.4%; n = 14), indicating that CFC
numbers increased proportionally to the total cell number. In 2 experiments, CAFC frequencies were also assessed. At day 0, these
frequencies were 5% and 14%, changing to 7% and 12% by day 4. These
numbers indicate that, as was the case with CFC, CAFC numbers increased
in proportion to total cell expansion.
In vitro assessment of gene transfer
Because the MGIN vector expresses the fluorescent marker EGFP, it
was possible to assess the efficiency of gene transfer into the
specific subpopulations of target cells using flow cytometry. Figure
2A illustrates the day 4 CD34/CD38
phenotype of cells in one experiment, with a gate defining
CD34+CD38 events. The percentage of
EGFP+ cells within the CD34+CD38
fraction (Figure 2B) was equal or slightly greater than that within the
total cell population (Figure 2C). Total cell gene transfer efficiency
averaged 20.1% (n = 13) and CD34+CD38
gene transfer efficiency, 20.8% (n = 11), as shown in Table
2. While 4 days in culture was
adopted as the standard infection protocol, we also examined the
efficiency of cell marking after 2 or 6 days. After 2 days (overnight
prestimulation and three 12-hour infections), the expression of EGFP in
the total cell population was very poor (mean, 2.4%; n = 2). This is
consistent with the observation of little or no expansion of cell
number by day 2 (Table 1), which suggests that few cells had entered mitosis by this time. At day 6, the efficiency of gene transfer into
"expanded" cells was somewhat greater than day 4 (26.5%; n = 1).
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| Fig 2.
Gene transfer into the "expanded"
CD34+CD38 subpopulation is comparable to
that into the total cell population.
(A) After 4 days of ex vivo transduction culture, cells were labeled
with anti-CD34-PerCP and anti-CD34-PE. R1 is a region defining the
CD34+CD38fraction. (B) A histogram shows
EGFP fluorescence in the total cell population. (C) A histogram shows
EGFP fluorescence within the CD34+CD38
fraction as defined by the R1 region shown in (A).
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CFC and CAFC assays were performed to test the efficiency of gene
transfer into clonogenic progenitor cells. As listed in Table 2, CFC
were transduced by day 4 at a higher frequency (mean, 26.3%; n = 12)
than that of total cells but also exhibited reduced transduction when
the culture period was shortened to 2 days. CAFC transduction after 4 days (3% and 8%; n = 2) was substantially less efficient than total
cell transduction. Intriguingly, in CAFC assays we were able to
reproducibly generate B-cell production in addition to the commonly
recognized myeloid cobblestone areas and hematopoietic areas (data not
shown). In rare cases, we observed EGFP marking of these B-lymphoid
cells, which are derived from a primitive lymphomyeloid
precursor.39
Ex vivo culture does not induce SRC expansion
While in vitro results indicated the dramatic expansion of cells
with a primitive (CD34+CD38) phenotype and
modest expansion of primitive cells with in vitro progenitor activity,
we were most interested in the fate of repopulating cells. We therefore
performed SRC assays in which NOD/SCID mice were injected with
uncultured or ex vivo transduced cells. The dose range was selected to
include 1 to 20 SRC, given that an average of 1.7% of uncultured
Lin CB cells are CD34+CD38
and that 1 in 617 of these cells is an SRC.9 Murine bone
marrow was assessed for human cell content after 6-8 weeks. Figure 3 shows the engraftment percentage of all injected mice, using values obtained by anti-CD45 flow cytometry in the case of well-engrafted (5% human cells) mice and by Southern blot quantification of all
others. By Poisson statistics, the SRC frequency of the uncultured cells was calculated to be approximately 1 in
2.5 × 105 ( 2 = 4.84). The SRC
frequency of cells following 4 days of transduction was 1 in
8.8 × 105 (2 = 7.58). When this
approximate 3.5-fold decline in SRC frequency is combined with an
average 4.2-fold increase in total cell number, the result is
maintenance but no significant expansionof SRC after 4 days in
culture. Thus, there was a dissociation between the
CD34+CD38 phenotype, which showed a dramatic
increase, and SRC, whose number remained approximately constant within
the same cultures.
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| Fig 3.
The engraftment levels of NOD/SCID recipients injected
with uncultured or ex vivo-transduced cells.
The percentages of human cells within the marrow of NOD/SCID mice
transplanted with uncultured Lin CB or ex
vivo-transduced Lin CB are shown. Values are derived
from flow cytometric analysis where engraftment exceeded 5% and
Southern blot quantification of all others. The dose of injected cells
is shown on the X axis. Closed circles represent mice containing
EGFP+ human cells, crossed circles indicate mice in which
the human cells did not contain EGFP, and open circles show mice that
could not be assessed for human cell transduction.
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Gene transfer into repopulating cells
The ex vivo transduction conditions described here were intended to
induce a significant percentage of SRC to enter mitosis, and therefore
becoming permissive to retroviral infection while retaining
repopulating activity. The human cells present in engrafted NOD/SCID
mice were therefore examined for evidence of gene marking by assessing
both EGFP fluorescence and the presence of G418-resistant CFC. Table
3 lists mice that were engrafted by
cultured cells and the extent to which their grafts contained
EGFP+ human cells. Of the assessed mice, 75% (12/16)
contained human CD45+EGFP+ cells at a level
1% (mean, 18%). The level of human cell marking was as high as 59%
in one instance. In the 11 mice that were characterized further, EGFP
expression was detected in both human B-lymphoid (CD45+CD19+) and myeloid
(CD45+CD33+) cells as well as CFC. Figure
4 provides a representative analysis of the
surface phenotype of human cells from a mouse containing a high
percentage of EGFP+ human cells. EGFP+ marking
was observed in myeloid (CD33) and lymphoid (CD19) lineages. Myeloid
and lymphoid marking was also present in 5 of 5 mice that contained
EGFP+ cells and that were injected with limiting doses of
transduced cells. This provides indirect evidence that a single marked
SRC can give rise to both myeloid and lymphoid progeny and is
consistent with our earlier data showing myeloid and lymphoid cells
present in mice transplanted at limiting doses. However, formal proof that the transduced myeloid and lymphoid cells shared a common precursor would require the demonstration of an identical retroviral integration site in both lineages.
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| Fig 4.
Both lymphoid and myeloid cells exhibit EGFP fluorescence
in mice engrafted with marked SRC.
(A) NOD/SCID marrow cells labeled with anti-CD45-PerCP and anti-CD19-PE
and analyzed by flow cytometry; region R1 defines the
CD45+CD19+ human B lymphocyte population. (B)
NOD/SCID marrow cells labeled with anti-CD45-PerCP and anti-CD33-PE;
region R2 defines the CD45+CD33+ human
myelocyte population. (C) EGFP fluorescence is detected within human B
lymphocytes using the R1 gate in (A). (D) EGFP fluorescence is detected
within human myelocytes using the R2 gate in (B).
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Characterization of CD34+CD38 cells
produced in culture
The combination of a large expansion of
CD34+CD38 cells without SRC expansion is
difficult to reconcile given the large body of evidence showing that
cells with this phenotype are highly enriched for primitive cells,
including SRC. To characterize these cells in more detail,
multiparameter flow cytometry was performed during 4 days of ex vivo
transduction culture assessing whether markers of maturation were also
present on the CD34+CD38 cells. Although a
proportion of the CD34+CD38 cells at the
start of culture expressed CD13 and CD33, the level of expression on
each cell was very low as monitored by the fluorescence channel. A
progressive increase in the frequency of
CD34+CD38 cells that also express high
cellular levels of CD13 (early myelo/monocytic marker) or CD33 (early
myeloid marker) was observed; by day 4, 95% and 93% of the
CD34+CD38 cells expressed CD13 and CD33,
respectively (Figure 5). Thus, the majority
of CD34+CD38 cells derived from cultured
cells appear to be fundamentally different compared with uncultured
cells because they show evidence of significant maturation.
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| Fig 5.
The frequency of CD13 and CD33 expression within the
CD34+CD38 fraction increases during ex
vivo culture (n = 2).
Uncultured (day 0) or ex vivo-cultured (days one to 4) cells were
labeled with anti-CD34-PerCP, anti-CD38-FITC, and one of anti-CD13-PE
or anti-CD33-PE, and analyzed by flow cytometry.
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Because a progressive decrease in the frequency of
CD34+CD38+ cells was observed while
CD34+CD38 cells were increasing in
frequency, we hypothesized that the expression of CD38 in relatively
mature CD34+CD38+ cells was subject to
downregulation during culture. Prior studies have shown that both
freshly isolated CD34+CD38+ cells and
CD34+CD38+ cells that are generated in ex vivo
culture from LinCD34+CD38
cells lack SRC activity.9,11 Therefore, the loss of CD38 expression on these cells would result in their classification as
CD34+CD38 diluting SRC activity in this
fraction. To assess this possibility, sorted
LinCD34+CD38+ cells were
cultured for 4 days under the same conditions as shown for Figure 1. In
addition, we wanted to examine the fate of isolated LinCD34-CD38 and
LinCD34+CD38 cells under
these conditions because all 3 fractions of cells are present within
the lineage-depleted samples used in our transduction experiments, and
both of these fractions have been shown to contain SRC.9,33
Figure 6 illustrates the phenotypic
assessment of each population in a representative experiment.
Interestingly, the phenotype of
Lin-CD34+CD38+ cells (Figure 6A)
changed dramatically after 4 days in culture (Figure 6B). By day 4, 23% of the cells adopted a CD34+CD38
phenotype while cell number increased by an average of 16-fold (n = 3). Therefore, approximately 4 × 105
"CD34+CD38" cells were produced after
an initial seeding of 1 × 105
CD34+CD38+ cells, representing a yield of 400%
in the CD34+CD38 fraction. When
1 × 105 sorted
LinCD34CD38 cells (Figure
6D) were cultured, approximately 5% acquired CD34 expression (Figure
6E) consistent with our recent studies33 (Bonnet et al, in
preparation); because the total cell number declined by an average of
2-fold in these cultures (n = 3), approximately 2.5 × 103 CD34+CD38
cells (a 2.5% yield) were present on day 4. Nearly all
LinCD34+CD38 cells (Figure
6G) retained the same phenotype while expanding by approximately
4.1-fold (410% yield; n = 3) after 4 days in culture (Figure 6H)
consistent with our earlier study11 under somewhat
different culture conditions. The values obtained in these experiments
with isolated subpopulations can be extrapolated to an unsorted
LinCB as follows. A typical uncultured
Lin CB sample of 1 × 106 cells
contains 5.13 × 105 (51.3%)
CD34+CD38+ cells, 1.6 × 105
(16%) CD34CD38 cells, and
1.7 × 104 (1.7%)
CD34+CD38 cells. With the yields calculated
above, after 4 days in culture the fraction that was originally
CD34+CD38+ should have generated
2 × 106
"CD34+CD38" cells, the original
CD34CD38 fraction should have produced
4 × 103
"CD34+CD38" cells, and the initial
CD34+CD38 fraction should have given rise to
7 × 104
"CD34+CD38" cells. Therefore, 96.4%
of the "CD34+CD38" cells present at
day 4 are predicted to have been derived from CD38 downregulation in
mature LinCD34+CD38+ cells that
contain no SRC activity.
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| Fig 6.
Sorted
LinCD34+CD38+ and
LinCD34CD38 cells can
become CD34+CD38 during ex vivo culture.
This representative experiment (n = 3) shows a LinCB
sample sorted to obtain CD34+CD38+ (A),
CD34CD38 (D), and
CD34+CD38 (G) fractions. These fractions
were individually cultured for 4 days in ex vivo expansion conditions.
Their CD34/CD38 phenotypes after 4 days in culture are shown in (B),
(E), and (H), respectively. Cultured cells labeled with PerCP- and
PE-conjugated mouse immunoglobulin G as isotype controls are shown in
(C), (F), and (I), respectively.
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Discussion |
In this report, we have developed conditions that result in both
efficient retroviral transduction of human SRC and their preservation
during short-term ex vivo culture. In addition, the use of the EGFP
marker gene together with flow cytometry enabled assessment of changes
in the cell surface phenotype of specific hematopoietic subfractions
during ex vivo culture and of the efficiency of gene transfer.
Unexpectedly, the ex vivo transduction cultures generated large numbers
of CD34+CD38 cells without a concomitant
increase in the number of SRC. This dissociation of the
CD34+CD38 cell surface phenotype, previously
associated exclusively with the stem cell compartment, and stem cell
function within the cultured cells indicates that caution should be
used when using flow cytometric methods to optimize stem cell
transduction and inferring stem cell function based on cell surface
phenotypes that are valid for uncultured cells.
In our earlier study, we concluded that SRC were relatively difficult
to transduce after finding no gene-marked cells in half of the
recipient mice and low levels (0.1%-3%) in the rest, even though all
mice contained high numbers of human cells. Only in rare (3 of 50) mice
could high levels (>20%) of marking be detected. In an attempt to
improve these results, many changes were made to the transduction
protocol. The conditions for cell culture were adapted from serum-free
conditions that we and others had earlier shown resulted in 2- to
4-fold expansion of SRC. The use of fibronectin as a cell support and
cell/virus colocalizer eliminated the need for a stromal layer and its
high serum requirements, although optimal virus production required the
presence of 1.5% FBS. A different retroviral vector (MSCV) was
employed that was designed for optimal expression within hematopoietic
cells. In addition, the retrovirus was pseudotyped with GALV rather
than the amphotropic envelope gene, because hematopoietic cells appear to express higher levels of this receptor. With the new protocol, EGFP+ human cells were found in 75% of engrafted mice and
up to 57% of the human cells were marked (mean 18%), indicating a
significant improvement in SRC transduction. Interestingly, half of the
mice containing marked human cells had been injected with a limiting dose of SRC. The myeloid and lymphoid cell marking in the human graft
of these mice indirectly suggests that the marked cells were derived
from a transduced SRC rather than 2 marked, lineage-committed precursor
cells. Recently, van Hennik et al22 used an EGFP-expressing retroviral vector to transduce SRC, and other groups have reported similar efficiency of SRC gene marking using a variety of different vectors and culture protocols.19-21,23 Together, these
studies provide strong support for new human clinical trials based on a
new generation of SRC transduction methodology.
Because mitosis is required for retroviral infection and the marked
human cells observed in engrafted mice are derived from transduced SRC,
it is reasonable to conclude that some of the SRC placed in ex vivo
transduction culture underwent selfrenewal divisions. Nevertheless,
no significant increase in the number of SRC occurred in culture. The
most significant difference between the transduction protocol reported
here and that of the expansion protocols of Bhatia et al11
and Conneally et al,12 who each observed a net expansion of
SRC, is likely to be the presence of 1.5% FBS throughout the protocol.
Serum is known to contain factors that promote cell differentiation,
and its exclusion may be a key element of the successful SRC
expansion protocols developed to date. Unfortunately, the withdrawal of
serum compromises the viability and virus production of retroviral
producer lines and thus may limit the efficiency of gene transfer. The
exclusion of serum by virus concentration40 or use of a
serum substitute21,41 has been shown to permit the
serum-free transduction of human hematopoietic cells. The results of
Schilz et al21 show that even in the absence of serum a
significant loss of SRC can occur. It is possible, however, that the
replacement of FBS with a serum substitute may enhance SRC survival;
efforts to explore this are in progress.
The retrovirus used in this study contained the EGFP gene, making it
convenient to monitor both the consequence of ex vivo culture on
various primitive cell fractions as well as gene marking of these
cells. Because our earlier studies had provided strong evidence for a
tight association between the
LinCD34+CD38 cell surface
phenotype and SRC function, it was anticipated that flow cytometric
analysis would provide a useful surrogate assay with which to optimize
transduction of the stem cell compartment in a manner less cumbersome
than long-term repopulation. Indeed, in these cultures
CD34+CD38 cells were readily marked with
EGFP. However, both the proportion and total number of cells with a
CD34+CD38 phenotype were dramatically
increased (166-fold, by number) with a concomitant reduction in
CD34+CD38+ cells. The combination of a large
increase in the number of CD34+CD38 cells in
culture and no increase in SRC number was surprising given that the
LinCD34+CD38 fraction is
known to be highly enriched for SRC activity. This suggested that the
CD34+CD38 cells that appeared after 4 days
were fundamentally different from uncultured cells with the same
phenotype, specifically with respect to SRC function. This idea was
supported by the demonstration that day 4 CD34+CD38 cells expressed high levels of
myeloid differentiation markers (Figure 5). Examination of the kinetics
of CD34 and CD38 expression in culture revealed a gradual decrease in
the proportion of CD34+CD38+ cells, resulting
in a "migration" of events from the
CD34+CD38+ quadrant to the
CD34+CD38 quadrant when assessed by flow
cytometry (Figure 1). This mechanism was confirmed by the emergence of
a substantial CD34+CD38 population when
sorted CD34+CD38+ cells are cultured under
identical conditions (Figures 6A and 6B). Quantitative analysis
suggests that most (96.4%) phenotypically CD34+CD38 cells present at day 4 are
actually derived from comparatively mature
"CD34+CD38+ cells" in which CD38
expression has been downregulated. Since neither uncultured
CD34+CD38+ cells nor those generated in
expansion cultures contain SRC,9,11 these cells almost
certainly contribute to the apparent "expansion" of
CD34+CD38 cells without providing additional
SRC activity. Similar to our earlier studies, the culture of
LinCD34+CD38 cells in the
transduction culture showed maintenance of the original phenotype,
suggesting that the maintenance of SRC in the original LinCD34+ cells used for the transduction
studies came from this fraction. It is also possible that at least some
of the SRC also were derived from
LinCD34CD38 cells and/or
the LinCD34+CD38 cells that
develop during 4 days of culture. In our earlier studies, SRC activity
was found in both cultured and uncultured
LinCD34 cells.33 As shown in
Figures 6D and 6E, a few CD34CD38 cells
are able to acquire CD34 expression in culture and appear as
CD34+CD38 cells on day 4. Because
CD34CD38 cells typically comprise
approximately 16% of a Lin CB sample and only 5% of
CD34CD38 cells are phenotypically
CD34+CD38 after 4 days (Figure 6E), the
frequency of SRC within these cells cannot be distinguished.
While, to our knowledge, the downregulation of CD38 on
Lin+CD34+CD38+ cells has not been
previously described, we note that in a flow cytometry analysis
published by Reems and Torok-Storb,42 a significant percentage of sorted CD34+CD38+ cells appear to
lose CD38 expression while retaining CD34 expression after 6 days in
culture. More recently, McCowage et al43 examined the
posttransduction phenotype of LinCB and showed that
under serum-free conditions a large expansion of
CD34hiCD38lo cells occurred. While repopulating
activity was not assayed in these experiments, the published
posttransduction CD34/CD38 flow cytometry data are similar to what we
have observed. Interestingly, the work of Rebel et al44
using primitive murine hematopoietic cells provides a striking parallel
with our own results. These researchers were able to expand primitive
murine LinSca-1+WGA+ cells by up
to 1000-fold in culture without any expansion of repopulating activity.
This phenomenon may be the result of a phenotype change, as with CD38,
or may reflect the selective expansion of a subset of
LinSca-1+WGA+ cells that lacks
repopulating activity.
It is unclear why CD38 should be downregulated on hematopoietic cells
during ex vivo culture. CD38 is a multifunctional membrane ectoenzyme
that is known to be downregulated as T-cell precursors mature from the
CD4+CD8+ stage into
CD4+CD8 or
CD4CD8+ T cells and when CD45RA+
T cells differentiate into CD45RO+ memory cells (reviewed
by Shubinsky and Schlesinger45). CD38 downregulation also
occurs during the maturation of B lymphocytes and has been shown to be
mediated by the activity of serine/threonine kinases during IL-4
signaling.46
As a functional test whose endpoint is the detection of engrafted human
cells, the SRC assay does not readily distinguish between the quality
and quantity of repopulating units. It is conceivable that the number
of SRC present in these culture conditions has in fact been expanded
but that a proportion of them has been impaired with respect to graft
durability, multipotentiality, or the capacity for homing to the
recipient bone marrow. With the identification of
LinCD34SRC, which can be functionally
distinguished from CD34+SRC, it has become clear that
multiple types of human SRC exist.33 The recent discovery
of the chemokine receptor CXCR4 as a candidate homing
molecule47 has revealed another level of regulation that should be amenable to experimental manipulation. The cytokines (IL-6
and SCF) that were shown to up-regulate CXCR4 expression by Peled et
al47 are present in the culture protocol described here,
but further investigation is needed to study the effects of ex vivo
expansion upon homing capacity.
In conclusion, we report the efficient transduction of SRC with a
retrovirus expressing EGFP. Combined with flow cytometric analysis of
CD34 and CD38 expression, this permits the monitoring of gene transfer
into cells of specific subpopulations. However, these results show that
the level of CD38 expression on cultured CD34+ cells is not
necessarily predictive of cell function. Our data show that the use of
a repopulation model such as the SRC assay is necessary to assess the
growth and differentiation behavior of stem cells in ex vivo culture.
| |
Acknowledgments |
We would like to thank Christine Botsford of Mount Sinai Hospital,
Toronto, Canada, for obtaining CB samples and Giselle Knowles, Department of Immunology, Hospital for Sick Children, Toronto, for
fluorescence-activated cell sorting of CB subfractions and Teresa
Hawley of The Toronto Hospital, Toronto, for generating PG13/MGIN
producer cells. We are also grateful to Kirin Brewery of Tokyo, Japan,
for the gift of its MS-5 stromal cell line.
| |
Footnotes |
Submitted November 12, 1998; accepted July 16, 1999.
Supported by grants from the Medical Research Council of Canada
(MRC), the National Cancer Institute of Canada (NCIC) with funds from
the Canadian Cancer Society, the Canadian Genetic Disease Network of
the National Centers of Excellence, the Blood Gene Therapy Program of
the Hospital for Sick Children, an MRC Scientist award, and an NCIC
postdoctoral fellowship.
Reprints: John E. Dick, Department of Genetics, Hospital for
Sick Children, 555 University Ave, Toronto, ON, Canada M5G 1X8.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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D.-W. Kim, Y.-J. Chung, T.-G. Kim, Y.-L. Kim, and I.-H. Oh
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Top
Abstract
Introduction