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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 1 (July 1), 1999:
pp. 106-113
Ex Vivo Expansion of Autologous Bone Marrow CD34+ Cells
With Porcine Microvascular Endothelial Cells Results in a Graft Capable
of Rescuing Lethally Irradiated Baboons
By
John E. Brandt,
Amelia M. Bartholomew,
Jeffrey D. Fortman,
Mary C. Nelson,
Edward Bruno,
Luci M. Chen,
Julius V. Turian,
Thomas A. Davis,
John P. Chute, and
Ronald Hoffman
From the Departments of Medicine and Radiation Oncology and the
Biological Resources Laboratory, University of Illinois at Chicago,
Chicago, IL; and the Naval Medical Research Institute, Bethesda, MD.
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ABSTRACT |
Hematopoietic stem cell (HSC) self-renewal in vitro has been
reported to result in a diminished proliferative capacity or acquisition of a homing defect that might compromise marrow
repopulation. Our group has demonstrated that human HSC expanded ex
vivo in the presence of porcine microvascular endothelial cells (PMVEC) retain the capacity to competitively repopulate human bone fragments implanted in severe combined immunodeficiency (SCID) mice. To further
test the marrow repopulating capacity of expanded stem cells, our
laboratory has established a myeloablative, fractionated total body
irradiation conditioning protocol for autologous marrow transplantation
in baboons. A control animal, which received no transplant, as well as
two animals, which received a suboptimal number of marrow mononuclear
cells, died 37, 43, and 59 days postirradiation, respectively.
Immunomagnetically selected CD34+ marrow cells from two
baboons were placed in PMVEC coculture with exogenous human cytokines.
After 10 days of expansion, the grafts represented a 14-fold to 22-fold
increase in cell number, a 4-fold to 5-fold expansion of
CD34+ cells, a 3-fold to 4-fold increase of
colony-forming unit-granulocyte-macrophage (CFU-GM), and a 12-fold to
17-fold increase of cobblestone area-forming cells (CAFC) over input.
Both baboons became transfusion independent by day 23 posttransplant
and achieved absolute neutrophil count (ANC) >500/µL by day 25 ± 1 and platelets >20,000/µL by day 29 ± 2. This
hematopoietic recovery was delayed in comparison to two animals that
received either a graft consisting of freshly isolated, unexpanded
CD34+ cells or 175 × 106/kg unfractionated
marrow mononuclear cells. Analysis of the proliferative status of cells
in PMVEC expansion cultures demonstrated that by 10 days, 99.8% of
CD34+ cells present in the cultures had undergone
cycling, and that the population of cells expressing a
CD34+ CD38 phenotype in the cultures was
also the result of active cell division. These data indicate that
isolated bone marrow CD34+ cells may undergo cell
division during ex vivo expansion in the presence of endothelial cells
to provide a graft capable of rescuing a myeloablated autologous host.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
THE SAFETY AND efficacy of ex
vivo-expanded human hematopoietic cell populations is already being
evaluated in the clinical setting.1-3 Currently, such
protocols are focused on the in vitro culture of large numbers of
mobilized peripheral blood CD34+ cells. Stem cell number
has been demonstrated to determine the marrow repopulating potential of
a graft.4-6 Ex vivo hematopoietic stem cell (HSC) expansion
is particularly important for cord blood transplantation. The potential
use of umbilical cord blood HSC as a source of grafts is limited by the
relatively low numbers of cells obtained, especially for an adult
recipient.7,8 The multitude of retroviral gene transfer
strategies currently being designed is also limited by the need for the
target HSC population to undergo cell division for integration of the
transgene to occur.9 Such cycling must occur in the absence
of differentiation for durable expression by cells derived from
modified stem cells to be possible. Several studies have shown,
however, that HSC lose their proliferative capacity after multiple
rounds of division, or that in vitro exposure to cytokines results in a
defect in homing or engraftment ability.10-15 Hematopoietic
cells comprising a graft must possess both the ability to home to the
marrow as well as retain a high proliferative capacity to reconstitute
hematopoiesis. The ideal ex vivo stem cell expansion system would thus
induce HSC to replicate in vitro without a loss of self-renewal
capacity or ability to home to the marrow.
In the absence of an appropriate human model, the baboon has proven
useful as a tool for the preclinical evaluation of HSC transplant
protocols.16-19 Several monoclonal antibodies, which recognize the human CD34 epitope, also cross-react with the analogous glycoprotein present on primate hematopoietic cells.16-20
CD34+ cells isolated from baboon bone marrow and mobilized
peripheral blood have been demonstrated to rescue lethally irradiated
animals and to restore lymphohematopoiesis.16,17,19 The in
vivo mobilization of baboon HSC and in vitro support of baboon
hematopoietic cells can be performed using readily available
recombinant human cytokines.18,20
Our group has recently reported the rapid in vitro expansion of adult
human marrow cells that retain a phenotype consistent with HSC when
cultured in the presence of a porcine microvascular endothelial cell
(PMVEC) line.21,22 These expanded HSC are capable of
engrafting and competitively repopulating human bone fragments
implanted in severe combined immunodeficiency (SCID) mice with
lymphoid, myeloid, and CD34+ progeny.21
Our goal was not to expand progenitor cell number, but rather to expand
the number of marrow repopulating cells by self-renewal and assess the
function of the expanded graft. In this report, we tested the ability
of the expansion product of autologous, selected CD34+
cells to engraft lethally irradiated baboons after 10 days of ex vivo
expansion culture in the presence of PMVEC and human cytokines. These
cocultures promote the in vitro cycling of HSC that remain capable of
reconstituting hematopoiesis in the host.
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MATERIALS AND METHODS |
Animals.
Healthy juvenile baboons (Papio anubis) of both sexes and
weighing 8.5 to 11 kg were used. The animals were housed under
conditions approved by the Association for the Assessment and
Accreditation of Laboratory Animal Care. The studies were performed
under protocols approved by the Animal Care Committee of the University
of Illinois at Chicago. Two weeks before transplant, the animals were
fitted with jackets and placed on a tether system; 1 week later,
central venous catheters were placed in the jugular and femoral veins. Beginning 4 days before transplant, the animals received eight fractions of 125 cGy total body irradiation (TBI) from a linear accelerator administered twice daily for a total of 1,000 cGy. After
completion of TBI (day 0), the animals were infused with a graft
composed of either mononuclear marrow cells, CD34+ marrow
cells, or the total cellular expansion product derived from marrow
CD34+ cells. The animals were administered 175 to 280 mL
whole irradiated blood transfusions from ABO compatible donors when
platelet counts fell below 20,000/µL or upon clinical evidence of
bleeding. Complete blood counts (CBC) were performed at 24- to 72-hour
intervals until the animals reached transfusion independence and
catheter removal; thereafter, CBCs were performed at weekly intervals
by blood collection under ketamine hydrochloride (HCL) (10 mg/kg) sedation. On reaching a neutropenic state defined as absolute neutrophil count (ANC) <500/µL, prophylactic antibiotics,
antivirals, and antifungals (ceftazidine 1,500 mg/d, gentamicin 100 mg/d, fluconazole 60 mg/d, acyclovir 100 mg/d, vancomycin 400 mg/d) were administered via continuous infusion until ANC >500/µL was achieved.
Collection, selection, and cryopreservation of
CD34+ marrow cells.
At least 6 weeks before transplantation, bone marrow aspirates were
obtained from the humeri and iliac crests of juvenile baboons after
ketamine and xylazine (1 mg/kg) anesthesia. Three sets of aspirates per
animal were obtained at intervals of at least 2 weeks to minimize
stress and the effects of loss of blood volume. The heparinized marrow
was diluted 1:15 in phosphate-buffered saline (PBS) and the mononuclear
cell fraction obtained by centrifugation over 60% Percoll (Pharmacia
LKB, Uppsala, Sweden) at 500g for 30 minutes. The monoclonal
antibody (MoAb) K6.1 (gift of the Naval Medical Research Institute,
Bethesda, MD), a murine IgG2a, which recognizes the
analogous baboon CD34 epitope, was used for the selection of the
CD34+ fraction of marrow cells.20,22 The
mononuclear cells were suspended in PBS containing 0.2% bovine serum
albumin (Sigma Chemical Co, St Louis, MO) and human immune globulin
(Bayer Corp, Elkhart, IN) and stained first with biotin-conjugated K6.1
(20 µg/mL), washed, and labeled with Miltenyi streptavidin-conjugated
iron microbeads (Miltenyi Biotech, Auburn, CA) and selected by passage through a magnetic column according to the manufacturer's
instructions. Purity of the positively and negatively selected cells
was determined flow cytometrically by counterstaining with
streptavidin-phycoerythrin (PE) (Southern Biotechnology, Birmingham,
AL). Purity of the CD34+ fractions was 93% to 98%. The
selection procedures were more than 99% efficient in enrichment of the
progenitor cells in the K6.1+ fraction as determined by
methylcellulose colony-forming cell assay and cobblestone area-forming
cells (CAFC) assays of the K6.1-positive and -negative cell fractions
(data not shown). The K6.1-selected or unselected mononuclear marrow
cells were cryopreserved at 2 × 107 cells/mL in 50%
Iscove's modified Dulbecco's medium (IMDM), 40% fetal
bovine serum (FBS), and 10% dimethyl sulfoxide (DMSO).
Ex vivo expansion cultures.
PMVEC were maintained and used for stem cell expansion cultures as
previously described in detail.21,22 Briefly, cryopreserved baboon K6.1+ cells were thawed and placed onto previously
established PMVEC monolayers in 162-cm2 flasks (Costar
Corp, Cambridge, MA) at 4 × 105 cells/mL in IMDM
containing 7% FBS and recombinant human stem cell factor
(SCF) at 100 ng/mL and interleukin-3 (IL-3), IL-6, and
granulocyte-macrophage colony-stimulating factor (GM-CSF) at 10 ng/mL
(gift of Amgen, Inc, Thousand Oaks, CA). At 3 to 4 days of culture, the
cocultures were dispersed by gentle agitation and half of the volumes
distributed to additional confluent PMVEC monolayers in
162-cm2 flasks and fed by replacement of fresh medium and
cytokines to the original volume in each flask. This procedure was
repeated at 3- to 4-day intervals to maintain a nonadherent cell
density of <2 × 106/mL. Mononuclear marrow cells
from an unrelated animal were also immunomagnetically depleted of
K6.1+ cells and the K6.1 fraction plated
in 35-mm tissue culture dishes (Costar) at 106 cells/well
in 2 mL IMDM containing 10% FBS and 2 × 105
mol/L 6 -methylprednisolone (Sigma). Cultures were fed weekly by
removal of all nonadherent cells and complete medium replacement weekly
for 4 weeks, by which time they were confluent with a population of
predominantly fibroblasts including adipocytes and macrophages.
Hematopoietic progenitor and CAFC.
Colony-forming units-granulocyte/macrophage (CFU-GM), burst-forming
units-erythroid (BFU-E), and colony-forming units-mixed lineage
(CFU-Mix) were assayed in methylcellulose culture as previously described.21 Briefly, 5 × 103
to 3 × 104 cells were plated in replicates of 1 mL
IMDM containing 1.1% methylcellulose, 30% FBS, 5 × 105 mol/L 2-mercaptoethanol, 100 ng SCF, 10 ng IL-3,
10 ng GM-CSF, and 5 U human recombinant erythropoietin (gift of Amgen,
Inc) in 35-mm tissue culture dishes (Costar). After 13 to 15 days of incubation at 37°C in a 100% humidified atmosphere of 5%
CO2 in air, the colonies were scored with an inverted
microscope using standard criteria for their identification. CAFC give
rise to uniform, multipotential aggregates of more than 50 uniformly
sized, refractile cells when cocultured for at least 5 weeks with
murine stromal fibroblasts in the presence of human cytokines. Marrow mononuclear cells, CD34+, or expanded cells were placed in
limiting dilution of 100 to 0.78 cells/well in 96-well plates onto
confluent, irradiated (7,000 cGy) monolayers of the murine stromal
fibroblast line M2-10B4. Each well contained 200 µL of a 50:50
mixture of IMDM and RPMI with 10% FBS, 50 ng/mL SCF, 50 ng/mL leukemia
inhibitory factor (LIF), and 5 ng/mL IL-3, IL-6, and GM-CSF, which has
been shown in our laboratory to optimize the development of CAFC. The
cultures were fed weekly by replacement of one half of the culture
volume with fresh medium containing the above cytokines at two
times final concentration. After 5 weeks of culture at
37°C in a 100% humidified atmosphere of 5% CO2 in
air, the CAFC-derived colonies were scored with an inverted microscope
using standard criteria for their identification.23 CAFC
frequency was computed using minimization of  by regression to the
cell number at which 37% of wells show negative CAFC growth, with 95%
statistical precision.
Proliferation analysis using PKH26 dye.
To ascertain the proliferative status of the CD34+
CD38 cells in PMVEC coculture, three baboon marrow
specimens were immunomagnetically selected for CD34+ cells
as described above. The CD34+ cells were next labeled with
the lipophilic membrane dye PKH-26 (Sigma), which fluoresces in the PE
channel, according to the manufacturer's instructions. The dye is
stably integrated into the cell membrane and the molecules are equally
distributed between daughter cells during cellular division; the
fluorescent intensity of each successive generation is thus half that
of the preceding one.21 The CD34+ marrow cells,
uniformly labeled with PKH-26, were placed into duplicate 35-mm tissue
culture dishes (Costar) containing preestablished monolayers of either
allogeneic baboon marrow stroma derived from the
K6.1 fraction of a fourth immunomagnetically
selected animal or PMVEC. At initiation and at days 7, 10, and 14 of
allogeneic stromal or PMVEC coculture, the cells were harvested and the
PKH-26 fluorescence of the CD34+ CD38
cells compared with that of the cells used to initiate the cocultures. CD38+ cells were stained with mIgG1 produced by
hybridoma OKT-10 (ATCC, Manassas, VA), which has been demonstrated to
cross-react with the baboon24 as well as
rhesus25 CD38 epitope, and labeled with fluorescein
isothiocyanate (FITC)-conjugated anti-mIgG1 (Southern Biotechnology). Harvested cells were stained with K6.1-biotin and
OKT-10 supernatant followed by streptavidin-allophycocyanin (APC) and
anti-mIgG1-FITC after two high-volume washes to remove any
residual serum. The cells were analyzed on a Becton Dickinson FACS
Vantage flow cytometer equipped with argon and helium/neon lasers
(Becton Dickinson, San Jose, CA). Nonviable cells were excluded by
detection of propidium iodide uptake. The flow cytometer was calibrated
with reference microbeads (Sigma), as well as FITC and APC Calibrite
reference beads (Becton Dickinson) and the photomultiplier tube voltage
and compensation settings saved for the subsequent analyses. Controls
consisted of cells stained with irrelevant mIgG1 followed
by anti-mIgG1-FITC (Southern Biotechnology) and irrelevant
IgG-biotin (Coulter Immunology, Hialeah, FL) and streptavidin-APC (Becton Dickinson). Greater than or equal to 3 × 104
cells were analyzed per culture at each time point.
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RESULTS |
Multiple bone marrow aspirates obtained from three baboons were
enriched for CD34+ cells using the MoAb K6.1 coupled with
immunomagnetic column selection and immediately cryopreserved. Ten days
before transplant, 8.1 × 106/kg (PA6156) and 8.4 × 106/kg (PA6188) CD34+ marrow cells were
thawed and placed into PMVEC cocultures, and expanded ex vivo for 10 days in the presence of exogenous human SCF, IL-3, IL-6, and GM-CSF.
This resulted in a fourfold to fivefold increase in the total number of
CD34+ cells and a threefold to fourfold increase in the
number of assayable CFU-GM after 10 days of PMVEC coculture
(Fig 1). Erythroid burst-forming cells were
also expanded in number by 9-fold to 17-fold (data not shown).
Primitive CAFC were expanded in number by greater than one log by day
10 of PMVEC coculture (Fig 1).
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| Fig 1.
Expansion of baboon CD34+ cells and
progenitor cells in PMVEC coculture. Bone marrow CD34+
cells from two baboons were immunomagnetically selected using the MoAb
K6.1 and used to inoculate 10-day expansion cultures in coculture with
the porcine microvascular endothelial cell line PMVEC in the presence
of exogenous human SCF, IL-3, IL-6, and GM-CSF. The total number of
CD34+ cells (dots), CFU-GM (vertical lines), and CAFC
(diagonal lines) were determined flow cytometrically, by
methylcellulose culture, and by limiting-dilution assay on murine
stromal fibroblasts, respectively, at inoculation (day 0) and at
harvest (day 10) immediately before infusion into the animals.
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To better understand the kinetics of primitive hematopoietic cell
expansion in the PMVEC cocultures, CD34+ cells were
selected from marrow specimens obtained from three baboons, labeled
with PKH-26, and inoculated onto either preestablished allogeneic
marrow stroma derived from the CD34 marrow cell
fraction of a fourth animal, or PMVEC monolayers. These cultures were
also supplemented with exogenous human SCF, IL-3, IL-6, and GM-CSF, and
the proliferation of CD34+ CD38 cells in
the cultures monitored using MoAbs K6.1 and OKT-10
(Fig 2). By day 10, the total number of
CD34+ cells in the PMVEC cocultures had increased to about
1.5 times the number used to inoculate the cultures, while
CD34+ cell numbers in the allogeneic stromal cell cultures
had declined to only 15% of input by day 7 (Fig 2A). By 7 days in
culture, 99.2% ± 0.3% of the CD34+ cells present in
the PMVEC cocultures had undergone multiple rounds of cell division
versus 84.7% ± 13.8% of the diminished number in the allogeneic
baboon stromal cell cultures; at day 10, the percentages of
PKH-26low were 99.8 ± 0.1 and 95.5 ± 4.5, respectively. These differences were reflected in the absolute number
of CD34+ PKH-26low cells present in the
cultures at 7 and 10 days, showing that the presence of
CD34+ cells in the cultures was due to proliferation rather
than survival of quiescent cells (Fig 2B). In contrast, the number of
cells in both culture systems expressing a primitive CD34+
CD38 phenotype decreased. CD34+
CD38 cells declined to an undetectable number in the
marrow stromal cell cultures (Fig 2C and
Fig 3), whereas significantly greater numbers of CD34+ CD38 cells were
maintained in the PMVEC cocultures after an initial decline (Fig 2C).
PKH-26 labeling confirmed that the presence of CD34+
CD38 cells in the PMVEC cocultures was also due to
proliferation of cells in this phenotypic compartment, where 99.25% ± 0.85% of the CD34+ CD38 cells
present in the cultures had become PKHlow by 10 days.
This contrasted with the proliferating CD34+ cells in the
allogeneic stromal cell cultures, which progressively gained CD38
expression (Fig 2D and Fig 3).
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| Fig 2.
Proliferation of CD34+ CD38
cells in allogeneic stromal and PMVEC coculture. Immunomagnetically
selected CD34+ baboon marrow cells were labeled with the
lipophilic dye PKH-26, which stably integrates into the cell membrane
and is distributed equally to daughter cells at division, and placed
onto preestablished allogeneic baboon marrow stromal cell (light bars)
or PMVEC monolayers (dark bars) in the presence of exogenous human SCF,
IL-3, IL-6, and GM-CSF. After 7 and 10 days of culture, CD34 and CD38
expression of the expanded cells was determined using the MoAbs K6.1
and OKT-10, respectively. Proliferative status of the cells was
determined by PKH fluorescence as described. The total numbers of
CD34+ cells (A), CD34+ cells, which have
undergone division in vitro (B), CD34+
CD38 cells (C), and CD34+
CD38 cells, which have undergone division (D) in the
cultures are shown. The data represent the mean ± standard deviation
(SD) of experiments performed on three separate animals. ND, not
detectable above 0.005% of events. *P < .05 (Student's
t-test);  P < .02; and  P < .001.
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| Fig 3.
Phenotype of baboon CD34+ bone marrow cells
after 10 days in allogeneic stromal and PMVEC coculture.
Immunomagnetically selected CD34+ baboon marrow cells (A)
were labeled with the lipophilic dye PKH-26 and placed onto
preestablished allogeneic baboon marrow stromal cell (B and C) or PMVEC
(D and E) monolayers as described in Fig 2. After 10 days of culture in
the presence of exogenous human SCF, IL-3, IL-6, and GM-CSF, the cells
were harvested and stained with the MoAbs K6.1 and OKT-10, which
recognize the baboon CD34 and CD38 antigens, respectively, and
secondarily labeled with APC (K6.1) and FITC (OKT-10). After first
gating on forward and side scatter and propidium iodide exclusion to
consider only live, nucleated cells, events positive for K6.1 (R3, top
row) were gated for OKT-10 versus PKH-26 analysis (bottom row). The
horizontal cursors define the PKH fluorescence of the freshly isolated
and labeled cells; events subsequently below this line have undergone
cell division. Machine settings were calibrated using reference
microbeads to assure consistency of PKH-26 measurement. The vertical
cursors delineate positive K6.1-APC (B and D) and OKT-10-FITC (C and E)
fluorescence based on both negative controls consisting of
isotype-matched irrelevant antibody secondarily labeled with APC or
FITC and positive fluorescence of reference beads; events to the right
of these cursors are positive. The data shown represent one of three
baboon marrows tested.
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Both animals, which were infused with the expansion product derived
from approximately 8 × 107 autologous
CD34+ cells (Table 1), became
transfusion independent within 23 days posttransplant
(Table 2). The pattern of hematological
reconstitution in the baboons is shown in
Fig 4. ANC >500/µL were reached by days
24 and 26, and stably increasing platelet counts >20,000/µL were
reached by 27 to 31 days posttransplant. These animals remained healthy
with normal blood counts (Table 2).
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| Fig 4.
Pattern of hematological reconstitution in the lethally
irradiated, transplanted baboons. Complete blood counts were performed
on the animals before irradiation to establish baseline values at 24- to 72-hour intervals after completion of myeloablation, and biweekly
after engraftment and removal from the tether system when applicable.
Differential counts of 100 nucleated peripheral blood cells, where
possible, were performed to determine the ANC. The initial 60-day
course of the seven animals' ANCs are depicted in the upper panel and
platelet counts in the lower panel. PA6216
() received no graft; PA6126
(- - - - - ) and PA6594 ( ) received a
suboptimal number of mononuclear marrow cells; PA6592 ( )
received a sufficient mononuclear marrow cell graft; PA6245 (--------- ) was infused with a sufficient number of selected, unexpanded
CD34+ marrow cells; PA6156 (----- )
and PA6188 ( ) were given grafts composed of the
cellular product of CD34+ marrow cells expanded for 10 days in PMVEC coculture.
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A third baboon was transplanted with 13.7 × 106/kg
unexpanded, cryopreserved, selected CD34+ marrow cells,
which were thawed, washed, and immediately infused after completion of
the TBI regimen (Table 1). This animal experienced a rapid recovery
with ANC >500/µL reached at day 16 and transfusion independence and
platelets >20,000/µL at 11 days after infusion of the graft (Table
1 and Fig 4). Although the baboon transplanted with an enriched
CD34+ cell graft received only 30% to 50% the number of
CD34+ cells, 50% to 70% the number of CFU-GM, and 20% to
50% of CAFC as the two animals that received expanded grafts (Table
2), its hematological recovery was more rapid and transfusion
independence was reached in half the time of that of the expanded graft
recipients. A control animal was infused with cryopreserved, unexpanded
whole mononuclear marrow with a similar total cell (175 × 106/kg) and CD34+ cell (21 × 106/kg) content to that of the two ex vivo-expanded grafts
(Table 1). This baboon also engrafted more rapidly than did the two animals that received the expanded grafts (Tables 1 and 2, Fig 4).
Three baboons were used as controls to determine the lethality of the
fractionated TBI regimen, as well as the potential of grafts containing
fewer than 4 × 106 CD34+ cells/kg to
rescue the irradiated hosts. A baboon that underwent the 4-day
irradiation schedule, but did not receive a graft, reached granulocyte
and platelet nadir by 7 days post-TBI (Fig 4). At this time, no
hematopoietic progenitor cells or CAFC were assayable from the severely
hypocellular marrow (data not shown). The animal died 37 days after the
myeloablative regimen (Table 2), by which time some granulocyte, but
not platelet recovery had occurred. A postmortem marrow sample
contained assayable CAFC, confirming that some degree of endogenous
myeloid recovery had occurred (data not shown). Two animals received
grafts composed of a suboptimal number of mononuclear marrow
cells16,17 (Table 1), which contained 1.0 and 3.8 × 106 CD34+ cells per kg of body weight. Both
remained pancytopenic with extremely hypocellular marrows until death
at 43 and 59 days posttransplant (Table 2 and Fig 4), at which time
insufficient marrow cells were obtainable for assay.
| |
DISCUSSION |
We have demonstrated the safety and effectiveness of rescuing
myeloablated nonhuman primates with expanded grafts derived from
CD34+ marrow cells. These studies show the ability of
microvascular endothelial cells to maintain the marrow engraftment
capacity of marrow-derived stem cells during cycling of the cells. It
is highly unlikely that the engraftment of the expanded marrow cells was due to the persistence of quiescent HSC which remain in
G0 and are thus more resistant to differentiation pressure
than actively dividing cells. At least 48 hours before harvest of the
PMVEC cocultures, greater than 99.5% of the CD34+ cells
present in the expansion cultures had undergone multiple rounds of cell
division as evidenced by membrane labeling; no subpopulation of cells
refractory to cycle induction was evident. The number of
CD34+ cells that remained quiescent in the cultures was
thus at least one log below the 1 to 3.8 × 106/kg
that were found inadequate for rescue of two myeloablated animals.
Because far greater numbers of native, unstimulated HSC were delivered
to the control animals, which received a suboptimal number of whole
mononuclear marrow cells,16 it is highly improbable that
the hematopoietic reconstitution observed in the animals receiving
expanded grafts was due to small numbers of HSC that failed to divide
over the 10 days of culture. PMVEC have been reported to drive the
extensive proliferation of highly enriched human HSC, which retain
their primitive CD34+CD38 phenotype
after several rounds of cell division.21,22 In these experiments, the numerical expansion of baboon CD34+ and
CD34+CD38 cells was lower than that
reported with human marrow cells by about one log. This discrepancy is
likely due to the administration of human recombinant SCF, IL-3, IL-6,
and GM-CSF to the cultures rather than recombinant baboon cytokines,
which are currently unavailable, resulting in suboptimal stimulation of
the baboon marrow cells. It is also possible that the signal(s)
produced by PMVEC that promote the expansion of the compartment of
cells possessing an HSC phenotype are not as efficiently transmitted to
the baboon cells, especially under culture conditions optimized for the
expansion of human cells. Here, PMVEC coculture has been demonstrated
to support the proliferation of baboon marrow cells expressing this
early phenotype in stark contrast to coculture with allogeneic baboon
marrow stroma. Such attenuation of the proliferation and generation of
progenitor cells by marrow stromal cells in the presence of multiple
cytokines has been reported.26
The expansions described herein were performed in the presence of
multiple human recombinant cytokines, specifically IL-3, IL-6, SCF, and
GM-CSF. Exposure of stem cell populations to certain cytokines, notably
IL-3, has been implicated in the acquisition of a defect in engraftment
capacity,10-15 especially in the absence of flt3
ligand.27,28 Recent studies of the engraftment of mobilized peripheral blood HSC in an autologous rhesus, as well as a
human/nonobese diabetic (NOD)/SCID xenogeneic system
indicate that the ex vivo expansion or even entry of mobilized
peripheral blood (MPB) HSC into cell cycle severely impairs their
ability to repopulate the host.29,30 Our group has reported
the correction of an apparent loss of marrow repopulation capacity by
adult human HSC expanded in the presence of IL-3, IL-6, and GM-CSF by
coculture with PMVEC in the absence of SCF and flt3
ligand.21 Whether prolonged in vitro exposure of expanded
grafts to the cytokines used in these experiments leads to an
impairment of long-term in vivo hematopoietic activity, which is
corrected by PMVEC coculture, remains to be tested.
The hematopoietic recovery of the two baboons transplanted with ex
vivo-expanded grafts was delayed in comparison to that experienced by
an animal that received a large number of unexpanded CD34+
cells or by an animal infused with a large number of unfractionated marrow cells. The animal transplanted with selected, unexpanded CD34+ cells received only one half to one third the number
of CFU-GM and one half to one fifth the number of CAFC as the two
baboons receiving the expanded grafts. Likewise, the expanded grafts, although initiated with fewer CD34+ cells than were
contained in the unexpanded grafts (8.1 to 8.4 × 106/kg v 13.7 to 21 × 106/kg),
contained two to three times the number of CD34+ cells
after expansion than did the selected, unexpanded or large mononuclear
marrow graft. However, although cells expanded for 10 days in PMVEC
coculture contained significant numbers of cells expressing a
CD34+CD38 phenotype that were the
product of cell proliferation, the total number of
CD34+CD38 cells was diminished from
input number, which may partially account for the observed delay in
engraftment by these cells. Güenechea et al31 have
recently reported the impaired short-term, but not long-term,
engraftment of human cord blood CD34+ cells expanded in the
presence of IL-3, IL-6, and SCF. Assayable progenitor cell content did
not correlate with rapidity of engraftment in these studies, and the
relevance of these in vitro data have been questioned of late in the
literature.29,31-33 These data and those of other groups
also suggest that the phenotypes of HSC and early progenitor cells may
deviate after prolonged in vitro culture from those of their native
counterparts, making their direct functional comparison to freshly
isolated cells by surface markers less
straightforward.21,34,35 Ongoing efforts to define
phenotypic and functional changes in HSC resulting from long-term in
vitro culture should prove useful in clarifying this controversy.
The level of TBI delivered to these animals has been generally accepted
to be myeloablative and lethal.19 While the regimen used
herein was certainly lethal, it is well recognized that no therapeutic
dose of radiation is totally myeloablative.36-38 Whether the expanded grafts are capable of providing durable, long-term hematopoiesis or simply rescue of the host until endogenous marrow recovery occurs is not possible to determine in the autologous stem
cell transplant setting without the complete and durable marking of HSC
and their progeny. Alternatively, sex-mismatched, compatible donors
have provided elegant tools for investigation of this
question.17 Such studies will be pursued to definitively establish the long-term contribution of the ex vivo expanded graft.
The safety of the delivery of marrow grafts expanded in culture in the
presence of xenogeneic stromal cells has been debated. The expanded
grafts used in this report contained some porcine cells, which were not
totally separated before infusion into the hosts. The engraftment of
foreign stromal cells harboring xenotropic viruses remains a legitimate
concern in the clinical setting. Indeed, porcine retroviruses and
endogenous proviruses capable of infecting human cells have been
identified in various porcine tissues.39 The persistence of
porcine tissue in the host and potential activation of porcine
retroviruses with tropism for human or primate cells will require
further investigation.
The data reported herein demonstrate the use of an ex vivo expanded HSC
product for hematopoietic reconstitution of a myeloablated primate
host. The rapid and total cycling of the population of cells used to
initiate these cultures suggests a pivotal role for PMVEC coculture in
inducing cell division without terminal differentiation for retroviral
gene marking and gene therapy protocols. Such approaches favoring stem
cell self-renewal in vitro may prove clinically useful for genetic
modification and the expansion of hematopoietic stem cell grafts.
| |
ACKNOWLEDGMENT |
The authors thank Christine Joy, Kimberly Gibbons, and Jeffrey Oswald,
DVM of the UIC Biological Resources Laboratory, without whose diligent
work this study would not have been possible.
| |
FOOTNOTES |
Submitted July 20, 1998; accepted March 2, 1999.
This work was performed under an evaluation Cooperative Research and
Development Agreement between the Naval Medical Research Institute and
the University of Illinois dated July 12, 1996. Views presented in this
manuscript are those of the authors and no endorsement by the
Department of the Navy has been given or should be inferred.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Ronald Hoffman, MD, University of Illinois
at Chicago, Section of Hematology/Oncology M/C 734, 900 S Ashland Ave,
Chicago, IL 60607.
| |
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ABSTRACT
INTRODUCTION