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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 4 (February 15), 1998:
pp. 1206-1215
Thrombopoietin, kit Ligand, and flk2/flt3 Ligand Together
Induce Increased Numbers of Primitive Hematopoietic Progenitors From
Human CD34+Thy-1+Lin Cells
With Preserved Ability to Engraft SCID-hu Bone
By
Karin M. Luens,
Marilyn A. Travis,
Ben P. Chen,
Beth L. Hill,
Roland Scollay, and
Lesley J. Murray
From SyStemix a Novartis Company, Cell Therapy Research, Palo Alto,
CA.
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ABSTRACT |
CD34+Thy-1+Lin cells are
enriched for primitive hematopoietic progenitor cells (PHP), as defined
by the cobblestone area-forming cell (CAFC) assay, and for bone marrow
(BM) repopulating hematopoietic stem cells (HSC), as defined by the in
vivo SCID-hu bone assay. We evaluated the effects of different cytokine
combinations on BM-derived PKH26-labeled
CD34+Thy-1+Lin cells in
6-day stroma-free cultures. Nearly all (>95%) of the CD34+Thy-1+Lin cells divided
by day 6 when cultured in thrombopoietin (TPO), c-kit ligand (KL), and
flk2/flt3 ligand (FL). The resulting CD34hi
PKHlo (postdivision) cell population retained a high CAFC
frequency, a mean 3.2-fold increase of CAFC numbers, as well as a
capacity for in vivo marrow repopulation similar to freshly isolated
CD34+Thy-1+Lin cells.
Initial cell division of the majority of cells occurred between day 2 and day 4, with minimal loss of CD34 and Thy-1 expression. In contrast,
cultures containing interleukin-3 (IL-3), IL-6, and leukemia inhibitory
factor contained a mean of 75% of undivided cells at day 6. These
CD34hi PKHhi cells retained a high frequency of
CAFC, whereas the small population of CD34hi
PKHlo postdivision cells contained a decreased frequency of
CAFC. These data suggest that use of a combination of TPO, KL, and FL
for short-term culture of
CD34+Thy-1+Lin cells
increases the number of postdivision PHP, measured as CAFC, while
preserving the capacity for in vivo engraftment.
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INTRODUCTION |
PLURIPOTENT hematopoietic stem cells
(HSC) are considered to be ideal targets for gene therapy. Use of
retroviral vectors for gene transduction requires that the target cells
pass through mitosis.1,2 Because the majority of freshly
isolated HSC are thought to be quiescent,3 it is necessary
to provide appropriate ex vivo conditions to stimulate HSC division
without differentiation and subsequent loss of multilineage potential
to achieve efficient clinical therapy with gene-manipulated HSC. Stroma
appears to be required to provide such conditions,4 but,
due to the technical difficulties of using stromal cultures for
clinical gene therapy trials, the appropriate culture conditions that
stimulate ex vivo replication of human HSC in the absence of stroma
must be defined.
To attempt retroviral gene transduction of HSC in the absence of
stroma, interleukin-3 (IL-3) and IL-6 in combination with c-kit ligand
(KL)4 or leukemia inhibitory factor (LIF)5 are
usually added to stroma-free cultures. However, the efficiency of gene
transduction into pluripotent HSC remains low. Ex vivo culture of HSC
with IL-3 can be detrimental to maintenance of primitive HSC function,
as was shown by decreased reconstituting ability of HSC in lethally
irradiated mice.6,7 Retrovirus-mediated gene expression in
human hematopoietic cells correlated inversely with growth factor
stimulation when cultures included IL-3.8 In addition, IL-3
can abrogate B-lymphoid potential and is a positive regulator of early
myelopoiesis.9 We have previously shown that 3 to 6 days of
culture in the presence of IL-3 induces not only cell division of
primitive human CD34+LinRhodamine
(Rh123)lo cells, but also differentiation (loss of CD34
expression).10,11 There is now increasing evidence that
inclusion of IL-3 in cultures results in loss of the long-term
reconstituting ability of HSC.6,7,12,13
We, therefore, wished to investigate combinations of early acting
stromal-derived cytokines for stimulation of
CD34+Thy-1+Lin cell division
without differentiation. Signaling through tyrosine kinase receptors
(TKR) is important to induce HSC division. The ligand for the TKR c-kit
(kit ligand) plays an important role in stimulation of proliferation of
HSC, usually in synergy with other cytokines.14,15 Another
important HSC-associated TKR, flk2/flt3, was first identified in the
mouse.16,17 Flk2/flt3 ligand stimulates proliferation of
both murine16,18-21 and human HSC.18,19,22-24
In addition to these two factors, thrombopoietin (TPO), although
originally believed to be a megakaryocyte (MK) lineage-specific cytokine,25 has been shown to stimulate
proliferation of HSC of both mouse26-28 and
human.10,11,29-31
The cobblestone area-forming cell (CAFC) assay allows in vitro
estimation of the frequency of primitive hematopoietic progenitor cells
(PHP) within a population, whereas the SCID-hu bone assay measures the
in vivo bone marrow (BM) repopulating ability of HSC. Both CAFC and
SCID-hu bone repopulating HSC are enriched among
CD34+Thy-1+Lin
cells.32-34 In the present study, we have compared
different cytokine combinations added to short-term cultures of adult
BM CD34+Thy-1+Lin cells for
retention of in vitro CAFC and in vivo SCID-hu bone repopulating
ability within the population of divided human CD34hi
cells. One condition used for gene transduction, ie, IL-3, IL-6, and
LIF, was compared with TPO plus KL11 and with TPO, KL, plus
flk2/flt3 ligand (FL).35 Newly generated CD34hi
cells could be identified by the loss of the fluorescent membrane dye
PKH26.36-38 The optimal timepoint for maximal division with minimal differentiation was determined. TPO, KL, and FL in combination were found to stimulate division of a majority of
CD34+Thy-1+Lin cells by day
4, with minimal loss of CD34 or Thy-1 expression.
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MATERIALS AND METHODS |
Antibodies.
To enrich for CD34+Thy-1+Lin
cells, we used Tuk3 (anti-CD34 obtained from Dr A. Ziegler, University
of Berlin, Berlin, Germany) directly conjugated to sulphorhodamine (SR)
and GM201 (antihuman Thy-1 from Dr W. Rettig, Ludwig Cancer Research
Institute, New York, NY) directly conjugated to phycoerythrin (PE;
SyStemix, Palo Alto, CA). As an isotype control for anti-CD34 (Tuk3)
staining, we obtained FLOPC 21 mouse IgG3 (Sigma, St Louis,
MO) conjugated to SR (SyStemix). As a control for anti-Thy-1 staining,
we used purified mouse IgG1 (Becton Dickinson, Mountain
View, CA) conjugated to PE (SyStemix). The lineage panel of fluorescein
isothiocyanate (FITC)-conjugated antibodies Leu-5b (anti-CD2), Leu-M3
(anti-CD14), Leu-M1 (anti-CD15), Leu-11a (anti-CD16), SJ25C1
(anti-CD19), FITC-conjugated mouse IgG1 and
IgG2a, PE- and FITC-conjugated HPCA-2 (anti-CD34), and
PE-conjugated Leu-12 (anti-CD19) and Leu-M9 (anti-CD33) were purchased
from Becton Dickinson. FITC-conjugated antibody D2.10 (antiglycophorin
A) was purchased from AMAC (Westbrook, ME). Hybridomas that produce
monoclonal antibodies to monomorphic or polymorphic determinants of HLA
molecules were obtained from American Type Culture Collection (ATCC;
Rockville, MD).39
Purification of
CD34+Thy-1+Lin
cells from BM.
Human adult BM (ABM) cells from normal donors were pre-enriched for
CD34+ cells using a magnetic bead selection device
(SyStemix). CD34+ cells were also selected from BM from two
multiorgan donors and frozen before use. CD34+ cells were
incubated for 10 minutes on ice with 2 mg/mL heat-inactivated human
gamma globulin (Gamimune; Miles Inc, Elkhart, IN) to block nonspecific
Fc binding. Subsequently, the cells were washed with staining buffer
(SB). SB contained Hank's Balanced Saline Solution (JRH Biosciences,
Lenexa, KS), 0.5% bovine serum albumin (Sigma), and 10 mmol/L HEPES
(Sigma). Cells were stained for 30 minutes on ice with anti-CD34-SR (6 µg/mL), anti-Thy-1-PE (10 µg/mL), and the lineage panel of
FITC-conjugated antibodies. Appropriate isotype controls were used, as
described above. Cells were then washed with SB and resuspended at a
concentration of 106/mL in SB containing 1 µg/mL
propidium iodide (PI; Molecular Probes Inc, Eugene, OR). A Vantage
fluorescence-activated cell sorter (FACS; Becton Dickinson
Immunocytometry Systems, San Jose, CA) was used to sort live
(PIlo)
CD34+Thy-1+Lin cells. The
sorts were reanalyzed to assure clean separation of cell
subpopulations.
PKH26 fluorescent dye labeling.
Cells were washed with protein-free PBS. The PKH26 dye (Sigma) was
diluted 1:250 in the kit diluent. The cell pellet was resuspended at a
concentration of 107/mL. This cell suspension was then
added to an equal volume of PKH26 and incubated for exactly 4 minutes
at room temperature (RT). An equal volume of fetal bovine serum (FBS;
Gemini BioProducts, Calabasas, CA) was then added and incubated for an
additional 1 minute at RT. An equal volume of Iscove's modified
Dulbecco's medium (IMDM) containing 10% FBS was then
added. The cells were counted and then centrifuged. The pellet was
resuspended at a concentration of 105/mL in IMDM/10% FBS
with and without cytokines for short-term suspension culture. The cells
were plated in round-bottom 96-well plates at 100 µL/well.
Short-term suspension culture.
PKH26-labeled cells were cultured for 6 days at 104
cells/100 µL of medium (IMDM, 10% FBS) in round-bottom 96-well
plates in suspension cultures containing different cytokine
combinations. The cytokines used included IL-3 (10 ng/mL), IL-6 (10 ng/mL), LIF (50 ng/mL; Novartis, Basel, Switzerland), TPO (10 to 15 ng/mL; R&D Systems, Minneapolis, MN), KL (50 to 75 ng/mL), and FL (50 to 75 ng/mL; SyStemix). Cell numbers were determined using a
hemocytometer and trypan blue to exclude dead cells.
FACS analysis of cultured cells.
A fraction of PKH26-labeled cells to be used as control was kept
overnight at 37°C in the absence of cytokines to remove unstably incorporated dye as well as antibodies bound to the surface and was
then stained with anti-CD34-FITC. The settings (PKH26 v
CD34-FITC) of the Vantage cell sorter were determined using these cells
that we called control day 0 (D0). At day 6 (D6), the wells were pooled and cells were counted and stained with anti-CD34-FITC antibody after
incubation with Gamimune. Cell division was measured by loss of PKH26
dye fluorescence and primitiveness by retention of the CD34 cell
surface marker (Fig 1).
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| Fig 1.
FACS analysis of
CD34+Thy-1+Lin cells
cultured for 6 days in various cytokine combinations containing FL.
Numbers are percentages of cells in each quadrant from a representative
experiment. Loss of PKH26 fluorescence indicates cell division. Loss of
CD34 expression indicates differentiation. Control cells were cultured
overnight (D0) or for 6 days (D6) without cytokines and quadrants were
set using live-gated undivided cells. The percentages of undivided cells (UR and UL quadrants) are combined.
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Undivided (PKHhi) and divided (PKHlo)
subpopulations of CD34hi cells were purified from 6-day
cultures containing IL-3, IL-6, and LIF or TPO and KL to determine if
PHP numbers were maintained or increased within the population of
CD34hi cells that had undergone division.
Figure 2 shows typical gates used for FACS
sorting from representative experiments. In each experiment, control
cells were cultured without cytokines and then stained with the
irrelevant mouse IgG1-FITC to set the gates. One example of
a control stain is shown for the TPO, KL, and FL combination. For this
cytokine combination, all cells were PKHlo and these were
divided into CD34hi and CD34lo/ subsets,
which were placed into the CAFC assay to determine the PHP frequency
and multilineage potential of the cells postdivision.
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| Fig 2.
FACS sort gates based on PKH26 versus CD34 fluorescence.
After 6 days of culture of
CD34+Thy-1+Lin cells in
different combinations of cytokines, cells were stained with
anti-CD34-FITC. Sort gates shown are on live (PI low) cells. These were
set based on the PKH26 profile of live unstimulated control cells. Each
cytokine condition required a different tissue for sorting and
therefore sort gates varied accordingly.
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CAFC assay.
A proportion of the cells was cultured at limiting dilution in the CAFC
assay as described previously.34 Briefly, cells were seeded
in 96-well plates preseeded with a murine stromal cell line (Sys-1) in
1:1 IMDM/RPMI medium (JRH BioSciences, Woodland, CA) containing 1 mmol/L sodium pyruvate (JRH BioSciences), 5 × 105 mol/L 2-mercaptoethanol (Sigma), and 10% FBS.
Limiting dilution ranged from 100 cells per well to 0.78 cells per
well. After 5 weeks, wells containing cobblestone areas were enumerated
and CAFC frequency of the cell population was calculated using maximum likelihood estimation with SAS software.40 The statistical
significance of CAFC frequency difference between cultured cell
populations was determined by ANOVA. Statistical significance of CAFC
number difference between cultured and starting cell populations was determined using the Student's t-test. Representative wells
containing cobblestone areas (at least 10 per sample group) were
individually analyzed by FACS for the presence of CD33+
immature myeloid, CD19+ B-lymphoid, and CD34+
progenitor cell populations to estimate the multilineage potential of
the original cells.
SCID-hu bone assay.
The SCID-hu bone assay was performed as previously
described.34,39 C.B-17 scid/scid mice were used as
recipients of human fetal bone grafts. First, limiting dilution
analysis was performed to determine the dose of
CD34+Thy-1+Lin cells that
reliably gives donor reconstitution in the SCID-hu bone model.
HLA-mismatched fetal bone grafts were injected with cell doses ranging
from 1,000 to 30,000 CD34+Thy-1+Lin cells per
graft into mice that received whole body irradiation (400 rad) shortly
before cell injection. To achieve a sufficient number of grafts at each
dose, four tissue donors were used in four separate experiments. Eight
weeks after injection, the bone grafts were recovered and the BM cells
harvested and analyzed for donor cell engraftment using FITC conjugates
of allotype-specific HLA antibodies versus PE-conjugated anti-CD19,
anti-CD33, and anti-CD34. Total human cells were detected with W6/32-PE
(antihuman HLA class I major histocompatibility complex
[MHC] molecule monomorphic determinant). Cells were
analyzed on a FACScan analyzer (Becton Dickinson Immunocytometry
Systems). Grafts having at least 1% of hematopoietic cells bearing
donor HLA antigen were considered positive. The percentage of grafts
showing donor reconstitution was assayed for each cell dose tested. At
five times the limit dose, or 10,000 cells, donor reconstitution was
observed in all grafts.
Uncultured BM CD34+Thy-1+Lin
as well as CD34hi PKHlo and
CD34lo/ PKHlo cells from D6 cultures in
TPO, KL, and FL were sorted and injected (10,000 cells per graft) into
SCID-hu bone grafts. Eight weeks after injection, the bone grafts were
analyzed for engraftment of donor CD33+, CD19+,
and CD34+ cells.
Kinetics of cell division.
A fraction of PKH26-labeled
CD34+Thy-1+Lin cells was
kept overnight at 37°C in the absence of cytokines and then stained
with anti-CD34-FITC. The settings (PKH26 v CD34-FITC) of the
FACS Calibur (Becton Dickinson Immunocytometry Systems) were determined
using these cells (control D0). Short-term suspension cultures of
PKH26-labeled CD34+Thy-1+Lin
cells were set up in different cytokine combinations, as described above. Cells were stained on D2, D4, and D6 with anti-CD34
(HPCA-2)-FITC and anti-Thy-1-Cy5 (GM201-Cy5 conjugated at SyStemix) and
analyzed on the FACS Calibur.
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RESULTS |
Increase of total cell number and of CD34+
cell number.
In the present study, we examined the effects of single, double, and
triple cytokine combinations in 6-day suspension cultures of
PKH26-labeled BM
CD34+Thy-1+Lin cells.
Previous studies using PKH26 did not show any detrimental effects of
PKH26 labeling on cellular function.36,38 As shown in
Table 1, single cytokines did not increase
the number of CD34+ or total cells. Combinations of two
cytokines of TPO, KL, and FL maintained the CD34+ cell
number with a slight increase (1.7-fold) in total cell number. Among
those tested, the combination of three cytokines, TPO, KL, and FL,
induced the highest increases of both total cell (4.7-fold) and of
CD34+ cell number (3.4-fold). The three-factor combination
IL-3, IL-6, and LIF did not stimulate an increase in total cell number.
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Table 1.
Comparison of Fold Increase of Total Cells and
CD34+ Cells in 6-Day Cultures in Single Cytokines or in
Cytokine Combinations
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Comparison of different cytokine combinations containing FL.
We examined the effect of FL alone and in combination with one or two
other cytokines in three to six experiments. In Fig 1, we demonstrate
how the quadrants were set on the control unstimulated cells and show
dot plots from a representative experiment. When cultured in FL alone,
most CD34+Thy-1+Lin cells
remained undivided (78%). Sixty-eight percent (mean 73%) of
postdivision cells lost CD34 expression. The addition of KL to FL
reduced by half the percentage of undivided cells (to 40%), and 55%
(mean 58%) of postdivision cells lost CD34 expression. The addition of
TPO to KL and FL stimulated much greater division (4% remained
undivided) with loss of CD34 on only 29% (mean 27%) of postdivision
cells. With other combinations containing TPO, eg, TPO and FL or TPO,
IL-3, and FL, we also observed that loss of CD34 expression only
occurred on about 30% of postdivision cells. We had previously shown
that IL-3 induces not only division, but also differentiation (CD34
loss) of human HSC.11 The addition of TPO seems not only to
contribute to greater cell division but also to overcome the effect of
IL-3 to promote differentiation.
To determine whether retention of CD34hi expression
postdivision correlated with retention of functional PHP, CD34/PKH26
subsets were purified postculture in three different cytokine
conditions and assayed for CAFC frequency. CD34hi
PKH26lo and CD34lo/ PKH26lo
subsets from TPO, KL, and FL cultures were also assayed for in vivo
SCID-hu bone repopulating activity.
FACS sorting of cultured cell subpopulations subdivided by PKH26
fluorescence and CD34 staining.
In subdividing CD34hi cells based on cell division, we
tried to exclude the CD34lo/ subpopulation, because
it is known that CAFC are contained mainly within the
CD34hi population,37 as confirmed in
Table 2. The majority of cells in IL-3,
IL-6, and LIF did not divide (mean 75%) by day 6; therefore, we sorted
CD34hi PKHhi versus CD34hi
PKHlo (mean 7.5%; Fig 2). The same cell populations were
sorted from cultures with TPO and KL in which a mean of 53% of cells
remained undivided and a mean of 23% of cells were CD34hi
PKHlo. In TPO, KL, and FL, all the cells had divided;
therefore, we sorted for CD34hi PKHlo (mean
71%) versus the CD34lo/ PKHlo (mean
26%) population of differentiated postdivision cells.
Analysis of CAFC frequencies and phenotype of cobblestone areas.
The PHP activity of the sorted CD34/PKH26 subpopulations of cultured
cells was estimated in vitro by use of the CAFC assay, comparing the
CAFC frequencies with the starting population of CD34+Thy-1+Lin cells. The
mean frequencies of CAFC within the starting population of
CD34+Thy-1+Lin cells ranged
from 1/21 to 1/46 (95% confidence limits, 1/16 to 1/52; Table 2).
Because of the limited number of cells obtained from each fresh BM,
only one cytokine combination could be tested per experiment, giving
rise to some tissue variation. In the case of IL-3, IL-6, and LIF, the
undivided CD34hi PKHhi subpopulation remained
primitive, retaining the same mean CAFC frequency as the preculture
CD34+Thy-1+Lin population.
However, the frequency of CAFC within the small CD34hi
PKHlo subpopulation had decreased 21-fold to a mean of
1/440 (1/249 to 1/813).
In addition, we evaluated the ability of cultured cell subpopulations
to give rise to both myeloid and B-lymphoid cells in long-term stromal
culture. Detection of CD34+ cells in 5-week cobblestone
areas suggests retention of primitiveness among the cell subpopulations
assayed. Analysis of a minimum of 10 small cobblestone areas generated
from this population showed that divided CD34hi cells from
cultures containing IL-3, IL-6, and LIF gave rise to only
CD33+ myeloid cells (Table 3).
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Table 3.
The Ability to Give Rise to B-Lymphoid and
CD34+ Progenitor Cells in Long-Term Stromal Cultures Was
Preserved for CD34hi Cells That Had Divided in TPO, KL,
and FL
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After culture with TPO and KL, the undivided CD34hi
PKHhi cells again had a similar CAFC frequency to the
uncultured CD34+Thy-1+Lin
population. In these conditions, the mean frequency of CAFC in the
divided CD34hi PKHlo subpopulation was reduced
2.3-fold, compared with the starting cell population (Table 2).
CD34hi PKHlo cells retained the potential, at
limiting dilution, to give rise to CD19+ B-lymphoid,
CD33+ myeloid, and CD34+ progenitor cells after
5 weeks of culture in the CAFC assay. However, the proportion of wells
containing greater than 1% CD34+ cells was reduced 22-fold
compared with the starting cell population (Table 3). For each cell
population, all cobblestone areas analyzed contained CD33+
myeloid cells.
The mean values for six experiments with the combination of TPO, KL,
and FL are shown in Table 2. The mean CAFC frequency remained the same
in the CD34hi PKHlo subpopulation, compared
with the starting
CD34+Thy-1+Lin cell
population. These cells also, at limiting dilution, retained their
ability to give rise to CD19+ B-lymphoid progenitors,
CD33+ myeloid cells, and CD34+ progenitor
cells. CD19+ cells were observed in a similar proportion
(~50%) of cobblestone areas examined for both the uncultured
CD34+Thy-1+Lin cells and
postculture CD34hi PKHlo cells. Forty-nine
percent of cobblestone areas generated from CD34hi
PKHlo cells contained CD34+ cells, as compared
with 67% for the uncultured
CD34+Thy-1+Lin cells (Table
3). As expected, the CD34lo/ PKHlo cells
had very low CAFC frequency (mean 1/3,000).
Increase in CAFC numbers among total and CD34hi
PKHlo cells.
We compared the increase of CAFC numbers from
CD34+Thy-1+Lin cells in
different culture conditions (Fig 3). Of
the three different cytokine combinations analyzed, only TPO, KL, and
FL increased the mean number of total cells, CD34+ cells,
and CAFC (Table 1 and Fig 3). If we compare the number of CAFC within
the CD34hi PKHlo population with the original
number of CAFC placed in culture, we can see that only in TPO, KL, and
FL were CAFC numbers increased among cells that had divided (mean
3.2-fold), although values ranged from maintenance to a 7.6-fold
increase. The CAFC number among divided CD34hi cells at day
6 was not significantly different from the number measured among
CD34+Thy-1+Lin cells at day
0 for TPO and KL cultures (n = 2, P = .19), but increased CAFC
number among CD34hi PKHlo cells in TPO, KL, and
FL cultures approached statistical significance (n = 6, P = .07). The number of measurable CAFC among divided CD34hi
cells from IL-3, IL-6, and LIF cultures had significantly decreased (n = 2, P = .03). All CAFC detectable in D6 IL-3, IL-6, and LIF cultures were derived from undivided CD34hi cells.
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| Fig 3.
Increase of postdivision CAFC numbers during 6 days of
culture in TPO, KL, and FL. Increased numbers of CAFC
(CD34hi PKHhi and PKHlo) were
determined by dividing the number at day 6 by the number placed in
culture at day 0 (left-hand columns). On the right, columns show the
fold increase in numbers of CAFC within the CD34hi
PKHlo (postdivision) population, compared with the number
within the CD34+Thy-1+Lin
population placed in culture at D0. There was a mean 3.2-fold increase
(range 1- to 7.6-fold) in postdivision CAFC in TPO, KL, and FL
cultures. Data shown for IL-3, IL-6, and LIF as well as TPO and KL are
the means of two experiments. Data for TPO, KL, and FL are the means of
six experiments (4 normal and 2 multi-organ donor BM). Error bars show
the SEM and P values indicate the significance of the change in
CAFC number from D0 to D6. ( ) IL-3, IL-6, and LIF; ( ) TPO and KL;
( ) TPO, KL, and FL.
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Dose of uncultured
CD34+Thy-1+Lin
cells that gives reconstitution of 100% of SCID-hu bone
grafts.34,39
The percentage of grafts showing donor reconstitution at each
CD34+Thy-1+Lin cell dose
tested is shown in Fig 4. Using Poisson
distribution analysis, the frequency of SCID-hu bone repopulating cells
was 1 per 2,000 CD34+Thy-1+Lin cells. At
five times this limit dose or 10,000 cells, donor reconstitution was
observed in 100% of grafts.
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| Fig 4.
Titration of BM
CD34+Thy-1+Lin cells in the
SCID-hu bone model. Bone grafts were injected with a range of doses of
CD34+Thy-1+ Lin cells (1,000 to 30,000) per graft. Data are the mean of four separate experiments
from 4 different BM donors. Donor reconstitution means that greater
than 1% of hematopoietic cells were positive for donor HLA antigen.
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Engraftment of CD34hi PKHlo cells
from 6-day culture in TPO, KL, and FL in SCID-hu bone.
CD34hi PKHlo cells from 6-day cultures of
CD34+Thy-1+ Lin cells in
TPO, KL, and FL clearly contained increased numbers of CAFC. In
addition, we asked whether the same cell population retained its
ability to repopulate human bone in vivo, using the SCID-hu bone
assay.34,39 To obtain sufficient cells, we purified
CD34+Thy-1+Lin cells from
cryopreserved BM CD34+ cells isolated from multiorgan
donors. Uncultured
CD34+Thy-1+Lin cells and
CD34hi PKHlo as well as
CD34lo/ PKHlo cells from D6 TPO, KL, and
FL cultures were injected into the fetal human bone grafts. Ten
thousand cells were injected per graft, because this cell dose provides
consistent engraftment of uncultured BM
CD34+Thy-1+Lin cells (Fig
4).
Cultured CD34hi PKHlo cells engrafted to a
similar level as the uncultured population of
CD34+Thy-1+Lin cells (4 of 4 grafts; Fig 5 and Table
4). In experiment A (Table 4), the mean percentage of
donor cells was 34.3% ± 22.3% for CD34hi
PKHlo cells, comparable with 25.0% ± 13.5% for
uncultured CD34+Thy-1+Lin
cells. FACScan analysis shows that multilineage engraftment occurred in
both cases, because the cells isolated from the bones after 8 weeks
included donor B-lymphoid (CD19+), myeloid
(CD33+), and progenitor cells (CD34+; Fig 5).
Cells of the CD34lo/ PKHlo subpopulation
engrafted in 1 of 4 bones and did not give rise to cobblestone areas in
vitro. This single engraftment (3.5% ± 5.3% donor) could have
been due to a low level of contamination (6%, seen in reanalysis) of
the CD34lo/ population with CD34hi
cells.
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| Fig 5.
CD34+ cells that have divided during 6 days
of culture in TPO, KL, and FL retain their capacity for in vivo marrow
repopulation in the SCID-hu bone assay. Uncultured
CD34+Thy-1+Lin BM cells and
CD34hi PKHlo and CD34lo/
PKHlo cells from D6 TPO, KL, and FL cultures were injected
into SCID-hu bone grafts (10,000 cells per graft). FACS analysis at 8 weeks showed multilineage marrow repopulation by both the uncultured cells and the CD34hi cells postdivision in culture
(PKHlo). The x-axis shows staining for donor HLA allotype.
The y-axis shows staining for total human cells (W6/32, antihuman class
I MHC) or lineage markers.
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View this table:
[in this window]
[in a new window]
|
Table 4.
CD34+ Cells That Have Divided During 6 Days of Culture in TPO, KL, and FL Retain Their Capacity for Marrow
Repopulation In Vivo in the SCID-hu Bone Assay
|
|
In a second experiment (B), in which the contamination of
CD34lo/ with CD34hi cells was less than
2%, we could show that 0 of 4 bones injected with the
CD34lo/ PKHlo subset engrafted, but 4 of
4 grafts injected with CD34hi PKHlo cells from
D6 TPO, KL, and FL cultures again showed multilineage engraftment, with
a mean of 59.0% ± 12.0% donor cells (Table 4). This confirms
that, after 6 days of culture, the in vivo marrow repopulating capacity
of CD34+Thy-1+Lin cells is
retained within the CD34hi population postdivision in TPO,
KL, and FL.
Comparison of the kinetics of cell division in TPO, KL, and FL and
IL-3, IL-6, and LIF.
For retroviral gene transduction of PHP and HSC, it will be important
to know the timepoint where division is maximal, but differentiation is
minimal. We, therefore, examined CD34 retention (primitiveness) and
PKH26 loss (division) by
CD34+Thy-1+Lin cells at D2,
D4, and D6. In addition, we stained the cells to follow Thy-1
expression as a marker of PHP (blue in Fig
6A). We chose to compare the cytokine combination of IL-3, IL-6, and
LIF with TPO, KL, and FL, which in our study stimulated greater
division of PHP with retention of primitive phenotype. A representative experiment (of 3 experiments) is shown in Fig 6.
View larger version (33K):
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[in a new window]
| Fig 6.
Kinetics of
CD34+Thy-1+Lin cell division
and differentiation during 6 days of culture. Comparison between IL-3,
IL-6, and LIF and TPO, KL, and FL. (A) PKH26 versus CD34 fluorescence.
Live Thy-1+ cells are shown in blue and live
Thy-1 cells are shown in red. Percentages of
PKHhi and CD34hi PKHlo and
CD34lo PKHlo/ cell subsets are shown for
this representative experiment (of 3 experiments). (B) forward versus
side scatter. Live Thy-1+ cells are shown in blue, and
live Thy-1 cells are shown in red.
|
|
In IL-3, IL-6, and LIF, 65% (mean 80%) of cells remained undivided at
D6. Forty-three percent of postdivision cells lost expression of CD34
and also appeared to lose Thy-1 expression (Fig 6A). Only 12% (mean
7%) of cells had divided by D4. In TPO, KL, and FL, most cells
underwent the first cell division between D2 and D4, because only 5%
(mean 7%) of cells lost PKH26 fluorescence by D2, yet already by D4,
73% (mean 75%) of the cells lost PKH26 fluorescence.
CD34hi expression was retained on 92% of postdivision
cells. By D6, 97% of the cells had divided with retention of
CD34hi expression on 76% of these cells. Clearly, Thy-1
expression (blue) was retained on the CD34hi
PKHlo cells from TPO, KL, and FL D6 cultures, which is
consistent with the retention of PHP and in vivo engraftment activity
demonstrated within this cell population. In Fig 6B, the early
myelopoietic effects of IL-3 could be detected as an increase in side
scatter of the cultured cells. In contrast, in TPO, KL, and FL there
was less cell death and the scatter profiles indicate a predominance of
blast morphology during the 6 days of suspension culture, consistent with less differentiation. The increase in size (FSC) of the
Thy-1+ cell subset in TPO, KL, and FL is shown in blue (Fig
6B).
| |
DISCUSSION |
TPO synergizes with KL to promote multilineage proliferation of both
human10,11,29,30 and mouse HSC.26-28,35 FL is a ligand for the flk2/flt3 tyrosine kinase receptor18,19 that seems to have a unique expanding effect on human peripheral blood long-term culture-initiating cells (LTC-IC).24 TPO has been shown to synergize with both KL and FL to enhance both the number and
size of clones formed by murine Sca1+
Lin progenitor cells.35 FL appears to
partially replace the requirement for stroma to maintain the human
long-term repopulating HSC during gene transduction,13
whereas IL-3, IL-6, and KL were insufficient.4
We have previously described the ability of TPO25 to
increase the number of human CD34+ cells detectable after
long-term culture.11 We also showed that TPO and KL could
synergize to drive division of primitive human BM
CD34+LinRhodamine123lo
cells with retention of CD34 expression.11 The question
remained whether the CD34+ cells that had undergone
division (PKHlo) retained primitive functional
characteristics. In the present study, culture with TPO, KL, and FL
stimulated virtually all
CD34+Thy-1+Lin cells to
divide by day 6, with a 3.4-fold increase in numbers of
CD34+ cells.
Expansion of LTC-IC (mean, 7.5-fold) has previously been described from
whole BM mononuclear cells using 14-day continuous perfusion culture
bioreactors containing a stromal layer.41 In addition,
Petzer et al42 have shown 30-fold expansion of LTC-IC
within 10 days, starting with a highly purified HSC population and
using a combination of 6 cytokines, including KL and FL. In our study,
we have used static cultures containing only 3 cytokines (TPO, KL, and
FL) and observed a mean 3.2-fold increase of CAFC numbers within 6 days
within a population that has been shown to be postdivision, based on
loss of PKH26 fluorescence. The minor population of CD34hi
PKHlo postdivision cells from cultures with IL-3, IL-6, and
LIF were found to contain very few CAFC, in contrast to undivided
CD34hi PKHhi cells, which retained CAFC at high
frequency.38 Only by using PKH26 to separate undivided and
divided CD34hi cells could we show that CAFC in cultures
containing IL-3, IL-6, and LIF represent undivided cells, whereas CAFC
from cultures with TPO, KL, and FL had all been generated de novo by
cell division. In the study by Petzer et al,42 only TPO and
FL when used alone stimulated a net increase of LTC-IC from
CD34+CD38 cells within 10 days. TPO and
FL have also been shown to induce extensive renewal with little
differentiation of cord blood LTC-IC ex vivo.31 In our
system, in TPO and KL or in TPO, KL, and FL, CD34hi
PKHlo cells showed retention of the ability to give rise to
B-lymphoid as well as early myeloid cells in 5-week stromal cultures.
Long-term bone repopulating cells are likely to be more primitive than
the majority of those that read out in the 5-week CAFC assay. Our
demonstration that CD34hi PKHlo cells from
6-day cultures with TPO, KL, and FL retained the ability to give a high
level of engraftment (both B-lymphoid and myeloid) at 8 weeks in our
SCID-hu bone transplant model indicates that the multipotency and
engraftment potential of
CD34+Thy-1+Lin cells was
preserved during cell division in vitro. Ex vivo expansion of HSC that
retains the ability to engraft may allow reduction of periods of
cytopenia when numbers of such cells are limiting for autologous
transplantation, as well as production of sufficient numbers to
overcome allogeneic transplant barriers.
Levels of gene transfer into pluripotent HSC remain low, potentially
due to the failure to induce division of the majority of primitive HSC
within the short transduction period. TPO has been proposed to shorten
the G0 period of dormant murine progenitor cells.27 We suggest that the rapid division of PHP
stimulated by TPO, KL, and FL may be due to this combination of factors
driving quiescent PHP to exit G0, as well as shortening the
G1 phase of the cell cycle.43,44 Based on these
premises, an optimal time to achieve integration of retroviral vectors
into dividing HSC would, therefore, be between day 2 and day 4, using
TPO, KL, and FL. Further studies will be necessary to determine whether
achieving maximal division of HSC will be sufficient to overcome the
barrier to transducing pluripotent long-term engrafting stem cells.
| |
FOOTNOTES |
Submitted June 17, 1997;
accepted October 6, 1997.
Address reprint requests to Lesley J. Murray, PhD, SyStemix, 3155 Porter Dr, Palo Alto, CA 94304.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely
to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors are grateful to all in the cell processing lab at SyStemix
for providing us with preselected BM CD34+ cells and to
Kwok Yu for conjugation of antibodies. We gratefully acknowledge
members of the SyStemix FACS Department, Brenda Lee, Jennine Lunetta,
and especially Mike Reitsma, for their support and helpful discussions
during cell sorting and analysis and Shirley Chen and Gun Hansteen for
SCID-hu bone assays. Thanks to Kathy Wright and the SyStemix protein
expression group for providing us with KL and FL, to Linda Osborne for
the Sys-1 cultures and help with CAFC frequency analysis, and to Chris
Gerard for consulting on statistical analysis. We also thank Dr Tim
Austin for critical review of the manuscript and Dr M. Abi Abitorabi
for valuable discussions.
| |
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Abstract
Introduction
Abstract