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

0851
CORD BLOOD DERIVED MESENCHYMAL STEM CELLS ARE EFFECTIVE AT PREVENTING
GRAFT-VERSUS-HOST DISEASE
V. Tisato, 1 K. Naresh, 1 J Girdlestone, 2 C Navarrete, 2 F Dazzi 3
1 Imperial College - Hammersith Campus, LONDON; 2 National Health Service
Blood and Transp, LONDON; 3 Stem Cell Biology Section, Kennedy Inst.,
LONDON, United Kingdom
Background. Evidence has emerged that Mesenchymal stem cells
(MSC) represent a promising population for cellular therapy and their
immunosuppressive properties make them particularly attractive to
manipulate graft-versus-host disease (GvHD). So far, the experience of
using MSC to treat GvHD is limited to a few cases and controversial
results come from preclinical models. Aim. The present studies were
designed to address these questions in a xenogenic model testing the
ability of Umbilical Cord Blood derived MSC (CB-MSC) to prevent and
/or treat GvHD. Methods. Subletally irradiatiated NOD/SCID mice transplanted
with human peripheral mononuclear cells (PBMC) selected for
their ability to engraft, showed extensive human T cells proliferation in
the peripheral blood, lymphoid and non lymphoid tissues, which
evolved in extensive GvHD (wasting, ruffled hair and hunched back).
Results. The chimeric-mice treated with a single dose of MSC did not
behave differently form the controls. However, when MSC were given
at weekly intervals, there was a marked decrease in human T cells proliferation
and none of the mice developed GvHD. No therapeutic effect
was obtained if MSC were administered at onset of GvHD. Conclusions.
This work supports the clinical use of MSC in SCT as a prophylaxis rather
than treatment of GvHD.
0852
FLOW-SORTED HUMAN HAEMATOPOIETIC STEM CELLS DO NOT TRANSDIFFERENTIATE
INTO FUNCTIONAL CARDIOMYOCYTES
K. Hensen, R. Konings, H. Jongen, M. Hendrikx, J.L. Rummens
Virga Jesse Hospital, HASSELT, Belgium
Background. Several clinical trials showed that purified CD133 + or
CD34 + cells injected in patiënts with myocardial infarction can contribute
to the repair of ischemic myocardium and improve heart function. The
mechanism responsible for this improvement in not clear yet and contradictory
reports have been published. Some groups declare that
haematopoietic stem cells (HSCs) are able to transdifferentiate into cardiomyocytes
(CMs), while others could not reproduce these findings.
Therefore further thorough investigations remain. Aims. This study aims
to examine haematopoietic stem cells when they are incorporated in a
cardiac environment. To mimic the microenvironment of the heart in vitro,
a co-culture system was developed in which flow-sorted human
haematopoietic stem cells were cultured in the presence of neonatal rat
cardiomyocytes (NRCMs). Methods. Mononuclear cells (MNC) were isolated
from human bone marrow (BM) samples. And incubated with
CD34-Pe-cy7 and CD133-PE antibodies. Subsequently, CD133 + /CD34 +
double positive cells were isolated under stringent purity conditions on
a FACSAria ® . Co-culture experiments were performed using celltracker
green (5-chloromethylfluorescein diacetate) labelled HSCs and celltracker
red (5-(and6)-(((4-chloromethyl)benzoyl) amino)tetramethylrhodamin)
labelled NRCMs. HSCs and NRCMs were plated at different
ratios and cultured in X-Vivo15 medium containing 2% fetal bovine
serum (FBS) or 2% autologous serum (AS) of the patient respectively.
Since several reports indicate that dimethylsulfoxide (DMSO) and 5azacytidin
(5-aza) induce myocardial differentiation, 1% DMSO for 48h
or 3 µM 5-aza for 24h was added and compared to conditions without
additives. After 3 weeks of incubation, green and red cell populations
were separated by flow-sorting and expression of cardiac specific genes,
including b-actin, Kv4.3, a-actinin, Connexin43, Troponin T, Troponin
I, a1c, Myosine Heavy Chain, GATA-4 Nkx2.5, were analysed by reverse
transcriptase polymerase chain reaction (RT-PCR). Results. Co-culturing
human HSCs with NRCM induced the expression of Troponin T while
expression of Connexin43 and Nkx2.5 was detected in both co-cultured
and freshly isolated HSCs. However, there was no expression of aactinin,
Myosin Heavy Chain, Kv4.3, a1C, Troponin I and GATA-4.
Adding DMSO or 5-aza had no influence on the differentiation of these
cells. Conclusions. Our results show no convincing evidence for transdifferentiation
of HSCs after 3 weeks of co-culture with NRCM. Even so,
no cell fusion between HSCs and NRCM could be detected. Probably,
other mechanisms like improved angiogenesis or paracrine effects stimulated
by HSCs can contribute to an improved heart function.
12 th Congress of the European Hematology Association
0853
PLASMACYTOID DENDRITIC CELLS AND TOLERANCE INDUCTION: IN VITRO ASSAYS ON
UMBILICAL CORD BLOOD HEMATOPOIETIC STEM CELLS
I. Varis, F. Pirlot, L. Di Pietrantonio, A. Friart, B. Brichard, D. Latinne
Cliniques Universitaires Saint Luc, BRUSSELS, Belgium
Background. The tolerizing function of both classic myeloid and more
recently identified plasmacytoid dendritic (pDC) cell subsets has become
obvious. They may be used as tools and targets to promote transplant
tolerance. There is growing understanding of the role of pDC in immune
tolerance in vitro by promoting differentiation of T regulatory (Treg) cells
and in vivo their administration may be effective in promoting T cell tolerance
in autoimmunity and transplantation. Aims. The aim of our study
is to produce and expand plasmacytoid dendritic cells from umbilical
cord blood (UCB) and to test their functional properties to promote in
vitro differentiation of T regulatory cells. Methods. CD34 + hematopo?etic
stem cells (HSC) were isolated from fresh human UCB by positive selection
with CD34 mAb coated beads and cultured with IL-3, Flt3-L and
SCF in ex vivo 20 medium (n=6) for 14 days. Phenotypical and morphological
analysis were performed at days 0, 3, 6, 9, 12 and 14 by MGG
staining and flow cytometry. The following markers were tested: CD2,
CD11c, CD19, CD22, CD33, CD34, CD45, CD56, CD64, CD123 and
CD304, specific markers for pDC. Results. Our culture system allows to
obtain until a 100 fold cell expansion at day 14. On MGG staining, cells
displayed a typical plasmacytoid cell morphology, characterized by an
excentric nucleus, a blue basophilic cytoplasm and pale Golgi zone. Cell
surface phenotype was analyzed by flow cytometry: the cells do not
express some lineages specific markers: CD19, CD22 (B cells), CD56
(natural killer cells), CD14 (monocyte), CD64 (FcgRI). At day 0, more
than 95% of cells were CD45 + CD34 + and CD33 + . At day 14, the number
of cells expressing CD34 decreased, a sign of cell culture differentiation.
A fraction of these cells expressed CD11c (23.55±10.2%), CD2
(10.1±6.4%), CD304 (35.5±9.5%) and CD40 (32.5±9.5%) but remained
CD123 negative. In two experiments, we activated the cultured cells
with soluble CD40L at day 14 and analysed the cells 24 h later. No significant
phenotypical differences were observed between activated and
non activated cells. However, soluble CD40L induced the development
of dendrites on plasmacytoid cells, a pDC characteristic described by
others. The observation that a fraction of cells generated from UCB
CD34+ cells in our culture system possessed morphological but not all
phenotypical characteristics of pDC, led us to assess their potential regulatory
function after activation by CD40L or CpG ODN A on proliferation
of allogenic naïve CD45RA + T cell. We evaluate by co-culture the
potential differentiation of naïve allogenic T cells into CD4 + CD25 + Treg
cells and their suppressor function on autologous and allogenic T cell proliferation
in vitro. These experiments are still in progress. Conclusions. We
were able to produce and expand plasmacytoid-like dendritic cells by culturing
UCB HSC in vitro with growth factors. These cells are morphologically
similar to pDC but they lack some markers in their phenotypic profile.
Co-cultures to test their potential functional immune regulatory
properties are in progress.
0854
A NOVEL SYSTEM FOR HIGHLY EFFICIENT CLINICAL SCALE PROPAGATION OF HUMAN
MESENCHYMAL STEM CELLS WITH HUMAN PLATELET LYSATE
K. Schallmoser, C. Bartmann, E. Rohde, A. Reinisch, K. Kashofer,
W. Emberger, G. Lanzer, W. Linkesch, D. Strunk
Medical University of Graz, GRAZ, Austria
Background. Human multipotent mesenchymal stromal cells (MSC)
are promising candidates for a growing spectrum of regenerative and
immune modulating cellular therapies. Translation of experimental
results into clinical applications has been limited by the dependence of
MSC propagation from fetal bovine serum (FBS). Aims. We analyzed the
capacity of human platelet lysate (HPL) to replace FBS for clinical scale
MSC propagation from human bone marrow (BM). Materials and Methods.
MSC expansion was performed under good manufacturing practice
conditions. Multiplex profiling was used to measure cytokines and
growth factors in HPL and compared to factor profiles derived from
expanded MSC. MSC function was further tested in potency assays for
clonality and differentiation. Genetic stability was determined using
conventional cytogenetics. Potential tumorigenicity of ex vivo expanded
MSC was studied in vivo by injecting graded MSC numbers into immune
incompetent mice. Results. HPL could be efficiently produced from normal
buffy coats. Multiplex analyses allowed delineating a distinct HPL
as compared to MSC-derived growth factor profile. Based on a previous-
haematologica/the hematology journal | 2007; 92(s1) | 317