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

12 th Congress of the European Hematology Association
lastoid stromal cell line (HFCL) on the proliferation, differentiation and
chemosensitivity of acute myeloid leukemia sensitive HL-60 cell line
and multidrug-resistant (MDR) HL-60/VCR cell line in vitro co-culture.
Methods. By setting up co-culture system of HL-60 or HL-60/VCR cells
in direct contact with HFCL cells, or with HFCL cells separated by transwell,
the cell growth curves were detected by cell counting, cell cycle
by flow cytometery(FCM). Cell differentiation was determined by morphologic
observation ability of NBT cells and flow cytometric detection
of expression of CD11b, CD14, CD13 and CD33.Exposing HL-60 or
HL60/VCR cells to different concentrations of topotecan(TPT), morphologic
evidence for apoptosis was determined by Wright-Giemsa and
Acridine Orange/ethidium bromide(AO/EB) staining. Cell cycle, Sub-
G1 and Annexin V FITC staining were detected by FCM. To further
study mechanism of HFCL cells on leukemic cells, we compared the
gene expression profiles of HL-60 cells without or in direct contact with
HFCL cells by Affymetrix GeneChip Human Genome U133 setA. The
expression of proliferation cell nucleus antigen(PCNA), active caspase-
3, bcl-2 and Pgp was detected by Western blot. VEGF levels were evaluated
by using commercial ELISA Kits. Results. Compared with leukemic
cells alone, the proliferation of HL-60 and HL-60/VCR cells cocultured
with HFCL cells was inhibited. And NBT positive cells increased slightly.
The percentage of G1 phase cells of HL-60 or HL-60/VCR cells cocultured
with HFCL cells was higher than that without HFCL cells, and that
of S phase cells was lower. The expression of CD11b and CD14
increased. The expression of PCNA was lower. HL-60 or HL60/VCR
cells treated by TPT were observed to have apoptosis characteristic morphological
changes by Wright-Giemsa and AO/EB staining. The percentage
of Annexin V-positive cells and apoptotic cells decreased when
they were cocultured with HFCL cells.The proportion of G0/G1 HL-60
or HL60/VCR cells treated with TPT increased and the sub-G1 was
33.43% or 21.9%, but sub-G1 reduced after in direct contact with HFCL
cells. In the study of mechanism, after direct contact with HFCL cells for
96h, the expression levels of 582 genes were up-regulated, and 1,323
genes were down-regulated at least twofold. The expression change in
some genes such as HL14, VEGF was comfirmed by RT-PCR, Northern
blot and ELISA, respectively. Meanwhile, with treatment with TPT in vitro,
the expression of activated caspase-3 was reduced and the expression
of bcl-2 increased in HL-60 or HL-60/VCR cells by co-culture of leukemic
cells in direct contact with HFCL cells. However, the expression of Pgp
showed no change. Conclusions. HFCL stromal cells could inhibit the
proliferation, induce the differentiation of HL-60 and HL-60/VCR cells,
and prevent TPT-induced apoptosis in HL-60 and HL-60/VCR cells via
modulation of Bcl-2 and active caspase-3. Many genes might take part
in the influence of HFCL cells on HL-60 cells, which may give important
insights into the interaction of bone marrow stromal cells and
leukemic cells.
1097
MESENCHYMAL STEM CELLS FROM UMBILICAL CORD VEIN, PLACENTA, AND BONE
MARROW DEMONSTRATE SIMILAR PHENOTYPE AND IMMUNOSUPPRESSIVE ACTIVITY
WHICH IS NOT MEDIATED BY APOPTOSIS
C. Barkatz, 1 I. Resnick, 2 D. Mancuta, 1 L. Weiss, 1 I. Reibstein, 1
B. Kurkalli, 1 G. Elkin, 1 L. Mizrachi, 1 S. Slavin, 1 O. Gurevitch, 1
I.B. Resnick1 1Hadassah, Hebrew Univ. Medical Center, JERUSALEM, Israel,
JERUSALEM, Israel
Introduction. Human mesenchymal stem progenitor cells (MSC) are
non-hematopoietic multipotent stem cells that are able to differentiate
along several pathways. MSC are widely distributed in a variety of tissues
in the adult human body; these cells are also present in the fetal
environment. MSC from different sources exhibit very similar but probably
not identical characteristics. in vitro and in vivo studies indicate
immunosuppressive activity of MSC. In the present study, our aim was
to compare phenotype and immunosuppressive activity of MSC from
umbilical cord vein (UC), adult bone marrow (BM) and placenta (P),
with a special focus on MSC of fetal origin. Methods. Ten placentas were
obtained from term deliveries after each mother signed a donation form
according to a protocol approved by the Research Ethics Committee of
our institution. The MSC from UC vein lumen or homogenized fetal
part of the placenta treated with collagenase, and cultured in DMEM
supplemented with 15% fetal bovine serum. BM-MSC used as a control.
Results. Initial results of our study demonstrate the following: 1)
MSC can be effectively isolated and grown both from placenta and
umbilical cord vein (7/10). MSC cultured from all three sources had
404 | haematologica/the hematology journal | 2007; 92(s1)
indistinguishable fibroblast-like morphology. 2) Similarly to BM-MSC,
UC-MSC and P-MSC exhibited characteristic immunophenotype with
the expression of CD90, CD166, HLA ABC, CD105, CD166, CD9 and
the lack of CD3, CD4, CD8, CD45, CD56, CD117, HLA DR, and Stro1.
3) Immuno-stimulatory activity of MSC was low, and UC-MSC caused
comparable mild activation of both allogeneic and autologous lymphocytes
(3.9±4.4 vs 2.7±1.5 fold higher cpm compared with controls, ND).
4) All three types of cells demonstrated uniform reduction of 3HT incorporation
by PHA-activated lymphocytes in range 40% to 90%
(p 10×10 9 /L and
platelets 10×10 9 /L, delayed achievement of molecular remission). Second remission
was achieved in 3 of them with treatment with As2O3, one pt is in
reinduction course now. With a median follow-up of 36 mo DFS is
0.80±0.09 and OS is 0.95±0.04. One pt died later in remission (accident).Conclusions.
Excellent DFS and OS were achieved in children and
adolescents with APL with treatment according the protocol with
chemotherapy with ATRA 25 mg/m 2 and reduced dose of anthracyclines.
Molecular monitoring of MRD is warranted during such treatment
of APL.