Le Blanc, K.

Rasmusson, I.

Ringden, O.

Sundberg, B.

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205
DEVELOPMENT OF A MINIMALLY INVASIVE BONE MARROW HARVEST
DEVICE AND METHOD FOR THE RAPID EXTRACTION OF BONE MAR-
ROW FOR USE IN BONE MARROW TRANSPLANTATION AND STEM CELL
THERAPY
Kraft, D.L., Milroy, C., Negrin, R.S. Stanford University School of
Medicine, Division of Bone Marrow Transplantation, Stanford, CA.
The bone marrow contains a rich supply of hematopoetic, mes-
enchymal and other stem cell populations. Bone marrow and pe-
ripheral blood–derived hematopoetic stem cells are currently used
in  40,000 bone marrow and peripheral blood stem cell trans-
plants each year worldwide. Adult bone marrow–derived stem cells
may soon be used in various stem cell–mediated regenenerative
therapies, resulting in an increased need for a simpliﬁed, econom-
ical acquisition of bone marrow. Allogeneic marrow, or G-CSF
mobilized marrow, may prove to be superior to PBSC for several
transplantation indications, and thus improved harvest methods are
needed.
Current methods of acquiring stem cells are tedious and expen-
sive. Traditional methods for harvesting bone marrow from pa-
tients are crude and generally require 100 or more separate inser-
tions of a large trocar needle into the donor’s ileac crest. Serial
trocar insertions allow aspiration of a small volume of bone marrow
each time, eventually obtaining a volume of marrow containing an
adequate number of stem cells. Marrow donors usually require
general anesthesia, multiple staff, and often an overnight hospital
stay. Peripheral blood stem cell harvests, alsthough less invasive,
require the donor to undergo several days of expensive G-CSF
administration, followed by many hours of apheresis. Traditional
bone marrow harvests and peripheral blood transplants incur av-
erage charges approaching $15,000 per harvest.
We have developed a novel harvest device, the MarrowMiner, for
rapid, minimally invasive extraction of bone marrow. This device
requires only 1 or 2 separate bone marrow entry sites, because after
entry into ileac crest, the device can access most of the accessible
marrow space. We anticipate that the MarrowMiner device will
enable a single operator to harvest bone marrow in a short period
with only local anesthesia required in the outpatient setting, sig-
niﬁcantly facilitating convenient, on- demand stem cell collection
while signiﬁcantly reducing harvest costs. Collected marrow could
be used immediately or undergo stem cell enrichment and manip-
ulation for various current and potential future therapeudic indi-
cations. Results from the MarrowMiner device’s preclinical devel-
opment in miniature swine and human cadavers will be presented.
HEMATOPOIESIS/MESENCHYMAL CELLS
206
IN VITRO UMBILICAL CORD BLOOD EXPANSION RESULTING IN
UNIQUE CD34BRIGHT CELL POPULATION THAT ENGRAFTS IN NOD/SCID
MICE
Xu, R., DeLuca, K., Porter, W., Garretson, L., Rowley, J.,
Liebmann-Vinson, A. BD Technologies, Research Triangle Parker, NC.
Umbilical cord blood (UCB) has gained recognition as a viable
alternative to bone marrow or mobilized peripheral blood for
hematopoietic stem cell transplantation and is being used for treat-
ment of mainly pediatric patients with various malignant or genetic
blood disorders. The limited number of cells contained in a single
cord blood unit remains the major bottleneck for broader applica-
tion of this treatment in adult patients. Extensive research is being
focused on strategies for overcoming this bottleneck. Expansion of
cells from a single cord blood unit or combining 2 cord blood units
for a single transplantation are the 2 most common strategies
currently in clinical evaluation.
Our research effort is focused on the expansion of umbilical cord
blood cells. At BD Technologies, we have developed a unique
higher-throughput technology platform that we have used success-
fully to identify a serum-free in vitro culture condition that sup-
ports expansion of UCB-derived cells. Expansion of UCB cells in
this condition results in a unique cell population, characterized by
a high level of glycoprotein CD34 expression. Characterization of
this expanded population using ﬂow cytometry, colony-forming
assays, and gene array analysis indicate that this expanded cell
population contains more primitive stem/progenitor cells than the
remainder of the CD34 population after expansion. When trans-
planted into sublethally irradiated NOD/SCID mice, these cells
were found to engraft in the bone marrow and to develop CD19
B-cell, CD33 myeloid, CD34 stem and progenitor, CD14
monocyte, and CD42 megakaryocyte lineages. Secondary trans-
plantation studies indicate the long-term engraftment potential of
the expanded cells.
In addition, when transplanted subcutaneously on porous poly-
mer carriers into NOD/SCID mice, the expanded cord blood cells
are found to initiate neovascularization within the carrier. Immu-
nohistochemistry revealed that human cord blood cells were in-
corporated into the new vasculature. We conclude that our culture
condition supports expansion of UCB cells that may have relevance
for therapeutic applications in humans.
207
BONE MARROW–DERIVED MESENCHYMAL CELLS DELAY ALLOGENEIC
REJECTION
Kleiner, G.I., Gandia, C., Yu, H., Kadono, J., Coats, E., Defaria, W.,
Santiago, S., Ruiz, P., Tzakis, A. University of Miami School of
Medicine, Miami, FL.
Bone marrow–derived mesenchymal stem cells (BM-MSCs) have
been demonstrated to have immunosuppressive effects in mice and
humans. These ﬁndings suggest that they may be exploited in
organ transplantation settings. We investigated immunoregulatory
aspects of rat BM-MSCs. Bone marrow was extracted from 6- to
8-week-old DA rats and plated in 10% complete medium. Every 48
hours, nonadherent cells were discarded. The immunosuppressive
effects of these cells in an allogeneic mixed lymphocyte reaction
were investigated. MSCs dramatically suppressed MLR response
(99%) when added at a concentration of 105 MSCs. Suppression
was dose-dependent. In ConA cultures, MSC suppression of lym-
phoid cells was maximal at 105 cells. MSC suppression was not
MHC-restricted, because Lewis-derived MSCs also suppressed
MLR and ConA responses ( 90%). Immunoregulatory levels of
IL-6, IL-10, and TNF-	 were detected in supernatant of MSCs
(BioPlex assay). The ability of MSCs to suppress allogeneic skin
grafts was evaluated in an allogeneic rat model. Allogeneic skin
grafts survived for 7 days without immunosuppressive therapy. In
preliminary experiments, MSCs (derived from DA) were injected
either as a single dose (10  106/kg) or as multiple doses given
every 2 days (on days 0, 3, and 5). MSC injections, either a single
injection or multiple injections, delayed skin graft rejection (days
8–12 or 14). Multiple MSC injections (10  106/kg) suppressed
intestinal transplantation rejection in a rodent heterotopic intesti-
nal transplantation model as well. The results suggest that MSCs
may have clinical utility in transplantation settings.
208
HUMAN MESENCHYMAL STEM CELLS AFFECT IGG PRODUCTION IN-
DUCED BY LIPOPOLYSACCHARIDE, CYTOMEGALOVIRUS AND VARI-
CELLA ZOSTER VIRUS IN HUMAN SPLEEN CELLS
Rasmusson, I.1, Sundberg, B.1, Le Blanc, K.1,2, Ringden, O.1,2 1Karo-
linska Institutet, Div of Clinical Immunology, Stockholm, Sweden; 2Cen-
ter for Allogeneic Stem Cell Transplantation, Karolinska University
Hospital Huddinge, Stockholm, Sweden.
Human mesenchymal stem cells (MSCs) suppress T-cell prolif-
eration and formation of cytotoxic T lymphocytes in vitro. In vivo,
MSCs prolong skin allograft survival and mitigate severe graft-
versus-host disease. The immunomodulatory properties of MSCs
have focused on T cells. We have examined the effects of MSCs on
human splenic B-cell IgG secretion. IgG response was analyzed
using the ELI-spot assays, after stimulating of human spleen cells
with lipopolysaccaride (LPS), cytomegalovirus (CMV), or varicella
zoster virus (VZV) antigens with or without 10% irradiated MSCs.
Unstimulated spleen cells gave 30  9 Spot-forming units (SFUs)/
Poster Session II
69BB&MT
100,000 cells, and after stimulation with LPS, it was 403 88 (n
28, adjusted to 100%). The addition of 10% irradiated human
MSCs gave 76  9%. Despite this suppression of IgG secretion,
we saw high variations in the experiments, as well as increased IgG
secretion. Stimulation with CMV gave a median of 330  98
SFU/100,000 cells (n  3), and the addition of 10% MSCs to the
culture reduced the response to 150  63. Stimulating spleen cells
with VZV gave 830 SFC/100,000 cells, which was reduced to 84
7 with 10% MSCs present. Enriched B cells were negatively
selected using immunomagnetic beads (Dynal, Norway). Using
enriched B cells, IgG production stimulated by LPS were variably
inhibited, unchanged, or stimulated after addition of 10% MSCs in
the culture, giving an average of increased IgG production (170 
53% compared with 100% in the positive control; n  5). Varia-
tions between different MSCs were similar to those using crude
spleen cells, indicating that MSCs most likely have a direct effect
on B cells, but because there might be some contaminating T cells
remaining in the culture (ﬂow cytometry showed  0.5% T cells),
we can not rule out that MSCs inhibit T cells and then in-direct
the B cells. When T cells are activated in mixed lymphocyte
cultures or by phytohemagglutinin, MSCs in high concentrations,
10% to 40% in the culture, mainly inhibit proliferation, whereas
low concentrations, 1%, in some experiments enhance lymphocyte
proliferation. For IgG secretion induced by LPS and viral antigens
CMV or VZV, 10% MSCs in culture inhibits IgG secretion in
most experiments, although a high variation in responses is seen
with this concentration. To conclude, MSCs inhibited B-cell IgG
secretion by human spleen cells induced by CMV and VZV.
However, induction by LPS on crude spleen cells or puriﬁed B
cells mainly stimulated IgG production.
209
DURABLE ENGRAFTMENT OF AMD3100-MOBILIZED HEMATOPOIETIC
STEM CELLS IN A CANINE AUTOLOGOUS AND ALLOGENEIC MODEL
Burroughs, L.1,2, Mielcarek, M.1,3, Little, M.-T.1, Bridger, G.4,
MacFarland, R.4, Fricker, S.4, Sandmaier, B.1,3, Torok-Storb, B.1,
Storb, R.1,3 1Fred Hutchinson Cancer Research Center, Seattle, WA;
2Department of Pediatrics, University of Washington, Seattle, WA;
3Department of Medicine, University of Washington, Seattle, WA;
4AnorMED, Inc., Langley, BC, Canada.
AMD3100 is a synthetic molecule that inhibits binding of stro-
mal cell–derived factor-1 to its receptor, CXCR4, expressed on
hematopoietic cells. AMD3100 treatment of animals or humans
results in mobilization of hematopoietic progenitor cells. Periph-
eral blood mononuclear cells (PBMCs) mobilized with a combina-
tion of AMD3100 plus G-CSF stably reconstituted autologous
hematopoiesis in cancer patients following myeloablative condi-
tioning. However, the engraftment potential of AMD3100-PBMC
used alone has remained unproven. We therefore studied this
potential in the autologous and DLA-identical littermate canine
hematopoietic cell transplantation (HCT) models. Pharmacoki-
netic studies performed in 4 dogs given a single dose of AMD3100
(4 mg/kg SC) demonstrated a 3- to 10-fold increase in CD34
cells measured by ﬂow cytometry, and a 2- to 5-fold increase in
CFU-GM and BFU-E within 8–10 hours of AMD3100 adminis-
tration. For autologous HCT, 4 dogs received a single dose each of
AMD3100 (4 mg/kg) followed by 3–4 hours of leukaphereses
initiated 6–7 hours after AMD3100 administration. Thereafter,
dogs were given a single dose of myeloablative total body irradia-
tion (TBI), 920 cGy at 7 cGy/min, followed by infusion of autol-
ogous AMD3100-PBMC (median CD34 cell count, 3.9 106/kg).
Neutrophil and platelet engraftment occurred at medians of 9 days
(range, 7–10 days) and 25 days (range, 23–38 days), respectively,
after HCT. At a median follow-up of 11.5 months (range, 8–12
months), all 4 dogs had normal marrow function. Because a con-
tribution of surviving endogenous stem cells to marrow function
could not be ruled out in the autologous HCT model, we next
evaluated the engraftment potential of AMD3100-PBMC in the
allogeneic HCT model. Four dogs were given AMD3100-PBMC
(median CD34 cell count, 3.4  106/kg) from DLA-identical
littermates after conditioning with 920 cGy TBI. Postgrafting
cyclosporine (5 mg/kg twice daily) was given from days1 to35.
Neutrophil and platelet engraftment occurred at medians of 9 days
(range, 8–10 days) and 26 days (range, 26–37 days), respectively.
At a median follow-up for living recipients of 162 days (range,
72–178 days), 3 dogs were alive and demonstrated 99%-100%
donor chimerism by VNTR analysis. One dog had early evidence
of engraftment but was euthanized 18 days after HCT because of
canine HSV infection and pancreatitis. In summary, both autolo-
gous and allogeneic AMD3100-PBMC transplants were associated
with timely and durable engraftment in dogs conditioned with
myeloablative TBI.
210
ALLOGENEIC T CELLS INDUCE CORD BLOOD CD34 OR CD133
CELLS RAPID PROLIFERATION AND DIFFERENTIATION INTO DEN-
DRITIC CELL PRECURSORS
Abbasian, J., Mahmud, N., Rondelli, D. Section of Hematology/Oncol-
ogy, University of Illinois at Chicago, Chicago, IL.
In this study we tested whether initial contact with allogeneic
lymphocytes may increase human cord blood (CB) hemopoietic
stem cells (HSCs) alloantigen-presenting capacity. Irradiated im-
munomagnetically puriﬁed CB CD34 and CD133 HSCs in-
duced similar proliferation (mean stimulation index, 19  5 and
16 5, respectively) of allo-responders in primary mixed leukocyte
culture (MLC). Cocultures of nonirradiated CD34 or CD133
cells with irradiated allogeneic T cells for 6 days resulted in a brisk
proliferation of HSC fractions (mean stimulation index, 17  2).
HSC proliferation was inhibited by B7:CD28 blockade with
CTLA-Ig. HSC that had been cocultured with irradiated alloge-
neic T cells for 6 days, were then rechallenged as stimulators in
MLC, and induced a signiﬁcantly greater T-cell alloresponse as
compared with the same freshly isolated HSCs (stimulation index,
134 54 vs 24 8) (P .03). The phenotypic kinetics of CD34
and CD133 CB cells on contact with irradiated T cells showed a
rapid up-regulation of CD86 within 24 hours (on average from 2%
 1% to 7%  2%, and from  1% to 4%  1%, respectively).
At 3 and 6 days of culture, both CD34 and CD133 CB cells
became larger and included a population of HLADR CD14
CD86 CD11c progenitors, on average 16%, consistent with
the phenotype of DC precursors. Also, irradiated allogeneic lym-
phocytes induced the generation of 8% CD1a DCs after 8 days
of culture with HSCs. To assess the effect of allogeneic lympho-
cytes on CB CD34 cell-derived DC generation in vivo, 10
NOD/SCID mice were irradiated (300 cGy) and injected with
0.20  106 CD34 CB cells and autologous (CD34/auto) or
allogeneic (CD34/allo) MNC (ratio 1:2), or with autologous or
allogeneic cells alone as control. At 6 weeks after transplant, mar-
row cells were harvested, and human CD45 cells were on average
1.8% in CD34/auto and 3.2% in CD34/allo. Although similar
proportions of B cells (CD45CD19) and monocytes
(CD45CD14) were detected in the two groups, a greater num-
ber of myeloid dendritic cells (CD45BDCA1) was observed
in CD34/allo than in CD34/auto (0.45%  0.30% vs 0.04% 
0.04%). These ﬁndings suggest that allogeneic lymphocytes may
induce a rapid differentiation of HSC into DC with potent alloan-
tigen-presenting capacity. Future studies aimed at either selectively
abrogating this subset of progenitors or blocking HSC:T-cell re-
ciprocal activation will be developed in the attempt of facilitating
the induction of tolerance in allogeneic HSC transplantation.
211
SUFFICIENT NUMBERS OF CORD BLOOD DERIVED MESENHYMAL STEM
CELLS FOR TRANSPLANTATION ARE GENERATED BY PLASTIC ADHE-
SION OR DEPLETING METHODS
Chatzistamatiou, T.K.1, Paterakis, G.2, Stavropoulos-Giokas, C.1,2,
Papassavas, A.C.1,2 1Hellenic Cord Blood Bank, Foundation for Biomed-
ical Research, Academy of Athens, Athens, Greece; 2Department of
Immunology & National Tissue Typing Center, General Hospital “G.
Gennimatas,” Athens, Greece.
Cord blood (CB) contains nonhematopoietic stem cells referred
to as mesenhymal stem cells (MSCs). These cells contained in CB
can be considered as “very young,” and thus CB may be an excel-
Poster Session II
70


Human mesenchymal stem cells affect IgG production induced by lipopolysaccharide, cytomegalovirus and varicella zoster virus in human spleen cells 

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Human mesenchymal stem cells affect IgG production induced by lipopolysaccharide, cytomegalovirus and varicella zoster virus in human spleen cells

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