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

12 th Congress of the European Hematology Association
ly established two-step clinical scale procedure, HPL was reproducibly
more efficient than FBS in supporting MSC outgrowth. Using only 3×10 5
MSC derived primary culture of less than 10 mL human BM we obtained
mean 4.36±0.51×10 8 MSC within a single secondary 11-13 day culture
step. Although morphologically distinct, HPL-MSC and FBS-MSC did
not differ significantly regarding immunophenotype, differentiation
potential in vitro and lack of tumorigenicity in nude mice in vivo. Conclusions.
A clinical quantity of functional human MSC for adult patients
could be reproducibly obtained from minute volumes of starting BM
under completely FBS-free conditions within less than four weeks.
Replacing FBS with HPL excludes bovine prion, viral and zoonose contamination
of the stem cell product. This new efficient FBS-free procedure
for clinical scale MSC propagation will largely facilitate rational
clinical testing of MSC based therapies.
0855
CLINICAL SCALE PROPAGATION OF FUNCTIONAL CORD BLOOD STEM CELLS IN A
HUMANIZED SYSTEM
A. Reinisch, C. Bartmann, E. Rohde, K. Schallmoser, V. Bjelic-Radisic,
G. Lanzer, W. Linkesch, D. Strunk
Medical University Graz, GRAZ, Austria
Background. Umbilical cord blood (UCB) is an easily accessible alternative
source for human hematopoietic stem cells (HSC) and mesenchymal
stromal cells (MSC). Limitations in UCB stem cell (SC) number have
so far hampered clinical applications of UCB for adult patients. Strict
dependence of SC expansion procedures from fetal bovine serum (FBS)
and inefficient expansion of HSC further limited clinical progress. Aims.
Expansion of cord blood stem cells in a FBS-free system for clinical applications.
Methods. We analyzed isolation and proliferation potential of
human UCB-MSC as compared to BM-MSC under optimized ex vivo culture
conditions. We further investigated the impact of human platelet
lysate (HPL) as an alternative to replace FBS for clinical scale expansion
of MSC. Progenitor cell function was determined by CFU-F assays and
correlated to proliferative senescence. MSC functions were tested in
hematopoiesis support, vascular regenerative and immune modulation
potency assays. Results. MSC cultures could be initiated from UCB with
and without FBS. MSC propagation was effective in 46% of UCB samples
compared to 100% of BM samples. Once established, the proliferation
kinetics of UCB-MSC did not differ significantly from that of BM-
MSC under optimized culture conditions resulting in > 50 population
doublings after only 10 weeks. A clinical quantity of 100 million MSC
could be obtained from UCB-MSC despite minute primary cell amounts.
Immune suppression and vascular regenerative function in vitro could
be shown for UCB-MSC propagated under both culture conditions. ex
vivo expansion of UCB-derived CD34 + /CD38 + hematopoietic progenitors
and CD34 + /CD38 – HSC was more efficient with HPL compared to FBScultured
UCB-MSC. Conclusions. We demonstrate for the first time that
human MSC can be obtained and propagated to a clinical quantity within
reasonable time from UCB in a completely FBS-free system. The efficient
propagation of UCB-derived CD34 + /CD38 – HSC encourages further
studies to develop effective strategies for UCB-SC expansion for
adult patients.
318 | haematologica/the hematology journal | 2007; 92(s1)
0856
THE ROLE OF WNT SIGNALING IN DIFFERENTIATION OF CORD BLOOD UNRESTRICTED
SOMATIC STEM CELLS INTO DOPAMINERGIC NEURONS
M. Mahmoodinia, 1 B. Zeynali, 1 M. Soleimani, 2 S. Kaviani, 2
A. Tafreshi3 1 University of Tehran,Faculty of Science, TEHRAN; 2 Department of Hematology,
Faculty of Med, TEHRAN; 3 Department of Biochemistry, National ins,
TEHRAN, Iran
Wnts are family of signaling glycoproteins that act through Wnt intracellular
transduction pathway transferring β-catenin, as a transcription
factor, from the cytoplasm into nucleus. There are large amounts of evidence
for the involvement of this pathway in early developmental
processes. Recent evidence shows that molecules in this pathway are
also expressed in mesenchymal stem cells and their niches. In this study,
we sought to examine the role of Wnt pathway in differentiation of
stem cells into dopaminergic neurons. Pluripotent cord blood stem cells,
known as unrestricted somatic stem cells (USSCs), were isolated from
mononuclear cells of the umbilical cord and recognized by specific markers
such asCD45, CD34. Neural differentiation factors such as basic
fibroblast growth factor and retinoic acid and a Wnt pathway inducer,
6-bromoindirubin-3-oxime (BIO), were used. Different dopaminergic
and neuronal markers such as TH, Nurr1 were considered for dopaminergic
neuron differentiation status of cells. Our results showed that in
the presence of both neural differentiation and Wnt inducer factors in
culture, more differentiated cells with morphological and molecular
characteristics of dopaminergic neurons were seen in comparison with
control cells. Moreover, there was a significant increase in the expression
of β-catenin in BIO treated cells, confirming that Wnt pathway is
activated in these cells. Our evidence on the involvement of Wnt signaling
in increasing dopaminergic differentiation of stem cells together with
its neuroprotective effect may have potential impact on therapy of neurodegenerative
diseases
0857
HUMAN PLATELET-DERIVED FACTORS INDUCE DIFFERENTIAL GENE EXPRESSION IN
BONE MARROW MESENCHYMAL STEM CELLS
C. Bartmann, 1 K. Schallmoser, 2 E. Rohde, 2 A. Reinisch, 3 C. Guelly, 4
G. Lanzer, 2 W. Linkesch, 3 D. Strunk3 1 Div. of Hematology; Transfusion Medicine, GRAZ; 2 Div. of Transfusion Medicine,
GRAZ; 3 Div. of Hemetology, GRAZ; 4 Center for Medical Research,
GRAZ, Austria
Background. The use of animal-derived products during human stem
cell processing bears the evident risk of xenogeneic prion, virus, or
zoonose contamination. Human platelet lysate (HPL) has recently been
recognized as a rich source of cytokines and growth factors with the
potential to replace fetal bovine serum (FBS) during ex vivo stem cell
manipulation. Aims. This study was performed to compare the gene
expression profile of human multipotent mesenchymal stromal/stem
cells (MSC) during ex vivo expansion for clinical applications under the
aegis of either FBS or HPL. Methods. The Applied Biosystems 1700
Expression Array System was used for full genome expression profiling
of MSC after a 12-14 day expansion period in a previously optimized
low density expansion system. Data have been obtained from biological
replicates. A starting amount of 40 µg total RNA was directly labeled
and DIG-labeled cDNA was hybridized to Human Genome Survey
Microarray V2.0. Attribution of regulated genes to biological processes
and pathways was done using the PANTHER ® db analysis software.
Results. Gene expression profiling identified a unique signature of human
MSC including 19 genes which discriminate MSC from mature fibroblasts
under both culture conditions. Interestingly, we identified 45 additional
genes that were more than two fold upregulated upon culture of
MSC in the humanized system compared to FBS supplemented media
(p