12th Congress of the European Hematology ... - Haematologica

ex-vivo expansion of HUCB-derived HSCs. The multidrug transporter
MDR1 (ABCB1) gene product (Pgp) and ABCG2 channel transporter
reported being over-expressed in various stem cells relatively to their
differentiated progeny. We, therefore, compared the expression level
and activity of Pgp in HUCB-derived CD133 + HSCs relatively to CD133 –
cells. Moreover, we reasoned that higher Pgp activity in these CD133 +
HSCs will make them more resistant to cytostatic agents as colchicine
(COL) and thus its presence during the expansion process could be applicable
to their selection and enrichment. Towards this end, we isolated
CD133 + HSCs from HUCB by CD133-immunomagnetic separation
(MACS). Pgp-expression level was measured by flow cytometry using
the Pgp-antibodies MRK-16, and its activity was measured by accumulation
assay of the Pgp-substrate Rh123. We further analyzed the
CD133 + /Pgp + and the CD34 + /CD38 – /Pgp + subsets during 8 weeks of standard
cytokines based expansion in the presence or the absence of COL.
Analyses of freshly isolated CD133 + HSCs from various donors (n=6)
indicated that the majority (>92%) of these HSCs express Pgp on the cell
surface. Moreover, the Pgp is functional as the accumulation of Rh123
was approximately 260-fold lower relatively to CD133 negative cells
and the Pgp inhibitor R-VRP inhibited the efflux of Rh123. Analyses
after ex vivo expansion demonstrated a significant dose-dependent
enrichment of CD133 + cell fraction by COL. At optimal COL dose (2.5
ng/mL, the relative fold-enrichment of CD133 + /CD34 + /CD38 – HSC was
5.2±2.3. At 8 weeks of expansion the CD133 + cell number increased
from 105 cells to 1.6±0.4×10 9 and 0.6±0.2×10 9 in the presence and
absence of COL, respectively and thus the total yield of CD133 + HSCs
after expansion in the presence of COL was 2.9±0.5 fold higher than in
its absence. The long exposure of CD133+ HSC to COL at the expansion
process did not affect their ability to form hematopoietic colonies
in semisolid cultures. In conclusion, we show that HUCB-derived HSCs
over-express a functional Pgp that may be used as a novel tool for their
expansion ex vivo towards clinical transplantation.
0449
PATIENT HSP70-HOM TG HAPLOTYPE IS ASSOCIATED WITH DECREASED TRANSPLANT-
RELATED MORTALITY AND IMPROVED SURVIVAL AFTER SIBLING HLA-MATCHED
HEMATOPOIETIC STEM CELL TRANSPLANTATION
I. Kim, 1 J.H. Kim, 2 J.Y. Rhee, 1 E.K. Ra, 1 S.C. Park, 1 E.K. Park, 1 B.S. Kim, 1
S.S. Yoon, 1 Y.C. Hong, 2 S.S. Park, 1 M.H. Park, 1 S. Park, 1 B.K. Kim1 1 2 Seoul National University Hospital, SEOUL; Seoul National University,
SEOUL, South-Korea
Background. Heat shock protein 70 hom (HSP70-hom) gene is known
to play an important role in protein folding and immune responses.
Therefore, HSP70-hom gene polymorphisms may act as important factors
predicting prognosis in patients receiving allogeneic hematopoietic
stem cell transplantation (HSCT).
Figure 1. Overall survival and treatment-related mortality according to TG
haplotype or non-TG haplotype.
12 th Congress of the European Hematology Association
Aims. The aim of this study is to evaluate the role of HSP70-hom gene
polymorphisms in prognosis of patients receiving sibling human leukocyte
antigen (HLA)-matched allogeneic HSCT.Methods. The HSP70-hom
polymorphisms, T2437C and G2763A, were genotyped in 147 patients
receiving sibling HLA-matched allogeneic HSCT, and individual diplotypes
were estimated from genotype data of two HSP70-hom polymorphisms
using the expectation maximization algorithm. Results. Patients
with 2763GG or GA genotype showed longer overall survival compared
with those with 2763AA genotype, and patients with TG haplotype
(TG/TA, TG/TG or TG/CG) also showed longer overall survival compared
with those without TG haplotype (TA/TA or TA/CG) (both
G2763A genotype and diplotype, p