Jones, M.

Levy, R.B.

Riley, R.L.

Shatry, A.M.

Van der Put, E.

Shatry, A.M

Van der Put, E

Riley, R.L

Jones, M

Levy, R.B

University of Miami: Scholarship Miami

Biology of Blood and Marrow Transplantation

A requirement for the spleen in preventing sustained imbalance of B220+ cells and GR-1+ cells during reconstitution following syngeneic and allogeneic BMT

Elsevier - Publisher Connector 

A requirement for the spleen in preventing sustained imbalance of B220+ cells and GR-1+ cells during reconstitution following syngeneic and allogeneic BMT 

HEMATOPOIESIS/MESENCHYMAL CELLS
176
NON-MYELOABLATIVE STEM CELL TRANSPLANTATION IN NEWLY ES-
TABLISHED DONOR STROMAL MICROENVIRONMENT FOR FACILITA-
TION OF HEMATOPOIETIC RECONSTITUTION
Slavin, S., Shapira, M.Y., Samuel, S., Kurkalli, B., Gurevitch, O., Or,
R. Hadassah-Hebrew University Medical Center, Jerusalem, Israel
Following allogeneic stem cell transplantation (SCT) migrate to
the hematopoietic microenvironment in bones, but the stroma
remains of host origin. We hypothesized that replacement of host
with donor stromal microenvironment supporting and regulating
hematopoiesis may enhance engraftment and differentiation of
bone marrow cells (BMC), especially in patients with myeloﬁbro-
sis. Our earlier work suggested that SCT with donor stromal
microenvironment modiﬁes GVHD. We have recently docu-
mented that transplantation of BMC mixed with demineralized
bone matrix (DBM) may be sufﬁcient for formation of new bone
and hematopoietic mircoenvironment. Tested in rats, implantation
of DBM/BMC composite intraosseously or under the kidney cap-
sule resulted in new bone formation containing donor hematopoi-
etic tissue. Based on the murine data, we introduced the use of
intra-osseous transplantation of DBM/BMC for patients with ex-
isting or anticipated marrow failure, such as myeloﬁbrosis, follow-
ing non-myeloablative conditioning (NST). Our 1st patient was 54
yrs old woman with RAEB-T and myeloid ﬁbrosis, with no hema-
tologic reconstitution following NST (ﬂudarabine, busulfex &
ATG) until week 4. Intraosseous (femora and pelvis) transplan-
tation of DBM/BMC was accomplished with no further condition-
ing from the same donor, assuming the problem was engraftment
failure rather than rejection. Early 3-lineage reconstitution of do-
nor (male) cells was conﬁrmed within one week. Currently, the
patient is more than 2 years out with mild chronic GVHD, with
100 percent normal male karyotype. Bone biopsy, laser capture
microdissection and PCR revealed 100 percent donor-derived
blood cells, stroma and bone cells. Two additional patients under-
went successful intra-osseous DBM/BMC transplantation into one
femur following NST, with fast 3-lineage engraftment. Procedures
were uneventful. All patients are disease free, with 100 percent
donor type cells. Considering our experimental and clinical expe-
rience, intra-osseous transplantation of DBM/BMC may provide a
new tool for enhancing allogeneic hematopoietic reconstitution,
especially for patients with myeloﬁbrosis.
177
ISOLATION OF OLIGODENDROCYTE PRECURSORS FROM UMBILICAL
CORD BLOOD
Hall, J.G., Crapnell, K.B., Staba, S., Kurtzberg, J. Duke University
Medical Center, Durham, NC
Introduction: The treatment of childhood leukodystrophies
with umbilical cord blood (UCB) transplantation has resulted in
cognitive and functional stabilization, but with minimal improve-
ment in neurologic and motor deﬁcits preexisting at the time of
transplant. The lack of improvement in motor function may be due
to the irreversible damage to motor neurons before transplanta-
tion. Conversely, trafﬁcking of transplanted cells may not occur for
months after transplant, missing a potential therapeutic window.
The delivery of cells directly to the brain may hasten engraftment
in and repair of neurologic tissue, facilitating clinical improvement
in these patients. We hypothesized that UCB contains cells capable
of differentiating into precursors of some neural lineages, speciﬁ-
cally oligodendrocytes, which could eventually be used for trans-
plantation in patients with these demyelinating or dismyelinating
diseases. To this end, we attempted to culture neural progenitor
cells from UCB ex vivo. Methods: Mononuclear cells were iso-
lated from fresh umbilical cord blood units with hespan and ﬁcoll
density gradient separation, and cultured in ﬂasks and LabTek®
chamber slides at a concentration of 3-4  106 cells/mL in media
containing platelet-derived growth factor (PDGF) (5ng/mL), neu-
rotrophin-3 (1ng/mL), tri-iodothyronine (30ng/mL), vascular en-
dothelial growth factor (VEGF) (10ng/mL), and 10% fetal calf
serum. An adherent cell population was observed after one week of
culture. Adherent cells grown on LabTek® slides were ﬁxed with
4% paraformaldehyde, stained and scored using immunocyto-
chemical techniques for the hematopoietic markers CD45 and
CD34, human leukocyte antigen (HLA) class I and II, microglial
markers CD68 and CD11b, the astrocyte marker GFAP, oligoden-
drocyte markers O1, O4, and myelin basic protein (MBP), and
neuronal markers NeuN and -tubulin III. Results: Approxi-
mately 60% of the isolated adherent cells appeared long and
spindle-like, many with branches extending from the cell body.
These cells displayed positive ﬂuorescent staining for CD45, O1,
O4, MBP, HLA I, and HLA II. The cells did not express CD34,
CD68, CD11b, GFAP, NeuN, or -tubulin III. Conclusions: The
pattern of staining observed in this UCB-derived cell population is
suggestive of oligodendrocyte differentiation. These cells may have
the potential for therapeutic trials in patients with leukodystrophies.
178
A REQUIREMENT FOR THE SPLEEN IN PREVENTING SUSTAINED IM-
BALANCE OF B220 CELLS AND GR-1 CELLS DURING RECONSTITU-
TION FOLLOWING SYNGENEIC AND ALLOGENEIC BMT
Shatry, A.M., Van der Put, E., Riley, R.L., Jones, M., Levy, R.B.
University of Miami Medical School, Miami, FL
The murine spleen is an active hematopoietic compartment fol-
lowing BMT and a target for preferred engraftment by a sub-
set (Rhohi) of hematopoietic stem cells (HSC). To determine the
contribution of the spleen to hematopoietic reconstitution after
BMT, the relative pattern of donor cell reconstitution and pro-
genitor cell (PC) distribution was examined in splenectomized
(Splx) recipients. Two weeks post-BMT, removal of the spleen
results in signiﬁcant increases in marrow PC activity compared to
sham-Splx controls in syngeneic and allogeneic BMT. Peripheral
and BM distribution of donor cells representative of lymphoid
(B220) and myeloid (Mac-1/Gr-1) cells was then assessed to
3 months post-BMT. A signiﬁcant (p  0.05) elevation of cir-
culating B220 cells was present in Splx recipients from 14 to 90
dys post-BMT, declining to sham control levels by 100 dys
post-BMT. In contrast, the % of circulating granulocytes (Gr-1/
Mac-1) was signiﬁcantly lower in Splx compared to sham con-
trols to 100 dys post-BMT. Three months post-BMT, the mar-
row compartment in Splx recipients demonstrated 3x (p 
0.0001) increases in sIgMD cells, although differences in cir-
culating B220 cells among the two groups had then diminished
by this time point, indicating a preferential accumulation of
sIgMD cells in this compartment. Initial results examining
LPS-stimulated PBMC proliferation are consistent with elevated
levels of circulating B220 cells (100 dys), indicating the B cells
are functional in Splx recipients. Additionally, consistent with el-
evated mature “recirculating” B cells in the marrow compartment,
LPS-induced proliferation levels were signiﬁcantly higher (p 
0.05) in these Splx recipients at 100 dys post-BMT. The in vitro
response of BM cells to IL-7 in Splx recipients was signiﬁcantly
(p  0.05) elevated, suggesting that the effects of the spleen on
B220 cells may be “upstream” of marrow sIgMD cells. A
decline in BM cell proliferation in response to IL-3 also occurred,
thus paralleling the observed peripheral decline in mature myeloid
cell subsets. These data indicate that the absence of the spleen in
BMT recipients could prompt sustained imbalance in the relative
distribution of deﬁned populations of the lymphoid and myeloid
lineages. Thus the splenic compartment is important in BMT both
as a site of hematopoiesis and in the regulation of deﬁned periph-
eral populations of the lymphoid/myeloid lineage.
179
COMPARISONS OF HEMATOPOIETIC SUPPORT BY ADIPOSE-DERIVED
AND BONE MARROW-DERIVED STROMA
Storms, R.W.1, Green, P.D.1, Foster, S.J.2, Gimble, J.M.2 1. Duke
University Medical Center, Durham, NC; 2. Artecel Sciences, Inc,
Durham, NC
Adipose-derived stromal (AdS) cells are multipotent mesodermal
progenitors with developmental potentials similar to those ascribed
Poster Session II
67BB&MT


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A requirement for the spleen in preventing sustained imbalance of B220+ cells and GR-1+ cells during reconstitution following syngeneic and allogeneic BMT

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